Peptide and peptide analog protease inhibitors

ABSTRACT

Methods of use of compounds and compounds for the treatment of disorders characterized by the cerebral deposition of amyloid are provided. Among the compounds are those of formulae I and II:    &lt;IMAGE&gt;  I   &lt;IMAGE&gt;  II  in which R1 is preferably 2-methyl propene, 2-butene, cyclohexyl or cyclohexylmethyl; R2, R4, and R8 are each independently methyl or ethyl; R3 is preferably iso-butyl or phenyl; R5 is preferably iso-butyl; R6 is H or methyl; R7-(Q)n is preferably benzyloxycarbonyl or acetyl; Q is preferably -C(O)-; R8 is preferbly iso-butyl; RA=-(T)m-(D)m-R1, is which T is preferably oxygen or carbon, and D is preferably a mono-unsaturated C3-4 alkenyl being more preferred; and X is preferably an  alpha -ketoester or  alpha -ketoamide.

This is a continuation of pending application Ser. No. 08/369,422, filedJan. 6, 1995.

FIELD OF THE INVENTION

This invention relates to peptidyl compounds useful for a variety ofphysiological end-use applications. More specifically, di- andtripepride analogs that are useful in the treatment of neurodegenerativedisease states and in the treatment of the degeneration of the neuronalcytoskeleton are provided.

SUMMARY OF THE INVENTION

Di- and tri-peptide compounds and methods of treating certain disorders,particularly cognitive disorders, and methods of inhibiting proteasesare provided. The methods use compounds having formulae: ##STR2## or thehydrates and isosteres, diastereomeric isomers and mixtures thereof, orpharmaceutically acceptable salts thereof in which:

R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R_(A), R_(B), X, Q and n are selectedfrom among (i), (ii), (iii), (iv), (v), (vi), (vii) or (viii) asfollows:

(i)

R₁, R₃, R₅, and R₈, are each independently selected from a side chain ofa naturally occurring α-amino acid, H, alkyl, preferably lower (C₁₋₆)alkyl, alkenyl, preferably C₂₋₁₀ alkenyl, alkynyl, preferably C₂₋₆alkynyl, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl,heteroaralkenyl, Y-substituted aryl, aralkyl, aralkenyl, aralkynyl, andZ-substituted heteroaryl, heteroaralkyl, heteroaralkenyl, in which Y isselected from halogen, lower alkyl, alkoxy, OH, haloalkyl, preferablyCF₃, NO₂, nitrile, S-alkyl, phenyl, and --NRR, R is H or alkyl,preferably lower alkyl, OH and halo-lower alkyl, particularly CF₃, Z islower alkyl, preferably C₁₋₄ alkyl, or halo lower alkyl, preferably C₁₋₄haloalkyl, more preferably CF₃ ;

R₂, R₄, R₆, and R₈ are each independently selected from among H andlower alkyl, preferably C₁₋₄ alkyl;

R₇ is selected from among C₁₋₆ alkyl, aryl, alkenyl, 9-fluoroenyl,aralkyl, aralkenyl, aralkynyl the aryl groups are unsubstituted or aresubstituted with Z;

Q is selected from among --C(O)--, --O--C(O), --S(O)₂ -- and HN--C(O)--;

n is zero or one;

R_(A) is --(T)_(m) --(D)_(m) --R₁ in which T is O or NH, and D is C₁₋₄alkyl or C₂₋₄ alkene; and m is zero or one;

X is selected from --(CH₂)_(r) C(O)H, --(CH₂)_(r) C(O)haloalkyl,--(CH₂)_(r) C(O)(CH₂)_(r) CHN₂, --C(CH₂)_(r) (O)C(CH₂)_(r) (O)OR_(D),

--(CH₂)_(r) C(O)(CH₂)_(r) C(O)NR_(D) R_(D), --(CH₂)_(r) C.tbd.N,--(CH₂)_(r) C(OH)(CH₂)_(r) C(O)U, --(CH₂)_(r) C(OH)CH₂ C(O)U,--(CH₂)_(r) C(O)W and --(CH₂)_(r) C(O)CH₂ W, in which: R_(D) is selectedfrom among H, alkyl, preferably lower alkyl, more preferably C₁₋₄ alkyl,phenyl, benzyl, and phenethyl; U is --OR_(D) or --NR_(D) R_(D), and W is--OR_(D), --SR_(D), and --NR_(D) R_(D), or heterocyclic moiety,preferably containing 4-6, more preferably 5 or 6 members in the ring,and preferably one or two heteroatoms, selected from O, S, or N, in thering, and r is 0-5, preferably 0-3, more preferably 0 or 1, mostpreferably 0; or

(ii)

R₁, R₂, R₃, R₄, R₅, R₈, X and Y are selected as in (i), (iv) or (v);

V is OH, halogen, lower alkyl, preferably methyl or ethyl orhalogen-substituted lower alkyl, preferably methyl or ethyl, and ispreferably preferably OH;

n is zero; and

R₆ and R₇ are each independently selected as follows:

(a) from lower alkyl, preferably C₁₋₃ alkyl, or lower alkyl linked to aheteroatom, preferably C₁₋₃ alkyl, with the proviso that there is atleast one carbon atom between the N to which R₆ and R₇ are each isattached and the heteroatom, and

(b) R₆ and R₇ are unsubstituted or substituted with one or moresubstituents selected from Y or preferably V, which is selected from OH,halogen lower alkyl, perferably methyl or ethyl, or halogen-substitutedlower alkyl, preferably methyl or ethyl, and is more preferably OH, and

(c) together with the atoms to which each is attached form aheterocyclic moiety, preferably a 5-6 atomed heterocyclic moiety, morepreferably selected from among morpholino, thiomorpholino, pyrrolidinyl,or V-substituted pyrrolidinyl, particularly 4-hydroxy pyrrolidinyl; or

(iii)

R₁, R₂, R₃, R₄, R₅, R₈, R_(A), X and R_(B) are selected as in (i);

V is as defined in (ii);

Q is C(O);

n is one; and

R₆ and R₇ are each independently selected as follows:

(a) from carbonyl (C═O), phenyl, a heteroatom, lower alkyl, preferablyC₁₋₃ alkyl, or lower alkyl linked to a heteroatom, preferably C₁₋₃alkyl, and

(b) each is unsubstituted or substituted with Y, preferably with V, and

(c) together with the atoms to which they are attached form a cyclicmoiety, preferably a 4-6 membered cyclic or 8-12 membered bicylicmoiety, and

(d) R₆ and R₇ are selected with the proviso that when two or moreheteroatoms are present there is a carbon atom between the heteroatoms;and

(e) when the moiety is a heterocycle, it is preferably succinimide,phthalimide or maleimide; or

(iv)

R₃, R₄, R₅, R₆, R₇, R_(A), R_(B), Q, X and n are as defined in any of(i)-(iii) or (v)-(viii),

V is as defined in (ii);

R₈ is H; and

R₁ and R₂ are each independently selected as follows:

(a) from lower alkyl, preferably C₁₋₄ alkyl, or lower alkyl linked to aheteroatom, preferably C₁₋₄ alkyl, or a heteroatom, with the provisowhen more than one heteroatom is present, there is at least one carbonatom between each heteroatom, and

(b) R₁ and R₂ are unsubstituted or substituted with Y, preferably withV, and

(c) together with the atoms to which they are attached form aheterocyclic moiety, preferably a 4-6 membered heterocyclic moiety, thatis preferably morpholino, thiomorpholino, pyrrolidinyl, or V-substitutedpyrrolidinyl, particularly 4-hydroxy pyrrolidinyl; or

(v)

R₁, R₂, R₅, R₆, R₇, R₈, R_(A), R_(B), X, Q and n are as defined in anyof (i)-(iv) or (vi)-(viii);

V is as defined in (ii);

R₃ and R₄ are each independently selected as follows:

(a) from lower alkyl, preferably C₁₋₄ alkyl, or lower alkyl linked to aheteroatom, preferably C₁₋₄ alkyl, or a heteroatom, with the provisowhen more than one heteroatom is present, there is at least one carbonatom between each heteroatom, and

(b) is unsubstituted or substituted with Y, preferably with V, and

(c) together with the atoms to which they are attached form aheterocyclic moiety, preferably a 4-6 membered heterocyclic moiety, thatis preferably morpholino, thiomorpholino, pyrrolidinyl, or V-substitutedpyrrolidinyl, particularly 4-hydroxy pyrrolidinyl; or

(vi)

R₁, R₂, R₃, R₄, R₇, R₈, Q, X and n are as defined in any of (i), (iv) or(v);

V is as defined in (ii);

R₅ and R₆ are each independently selected as follows:

(a) from lower alkyl, preferably C₁₋₄ alkyl, or lower alkyl linked to aheteroatom, preferably C₁₋₄ alkyl, or a heteroatom, with the provisothat when more than one heteroatom is present, there is at least onecarbon atom between each heteroatom, and

(b) R₅ and R₆ are unsubstituted or substituted with Y, preferably withV, and

(c) together with the atoms to which they are attached form aheterocyclic moiety, preferably a 4-6 membered heterocyclic moiety, thatis preferably morpholino, thiomorpholino, pyrrolidinyl, or V-substitutedpyrrolidinyl, particularly 4-hydroxy pyrrolidinyl; or

(vii)

R₁, R₂, R₃, R₄, R₆, R₈, R_(A), R_(B) and X are selected as in (i) (iv)or (v);

V is as defined in (ii);

n is zero; and

R₅ and R₇ are each independently selected as follows:

(a) from lower alkyl, preferably C₁₋₄ alkyl, or lower alkyl linked to aheteroatom, preferably C₁₋₄ alkyl, or a heteroatom, with the provisothat when more than one heteroatom is present, there is at least onecarbon atom between each heteroatom, and

(b) R₅ and R₇ are unsubstituted or substituted with Y, preferably withV, and

(c) together with the atoms to which they are attached form aheterocyclic moiety, preferably a 4-6 membered heterocyclic moiety, thatis preferably morpholino, thiomorpholino, pyrrolidinyl, or V-substitutedpyrrolidinyl, particularly 4-hydroxy pyrrolidinyl; or

(viii)

R₁, R₂, R₃, R₄, R₅, R₈, X and Y are selected as in (i), (iv) or (v);

V is as defined in (ii);

R₆ and R₇, which are defined as in (ii), together with the atoms towhich each is attached form a bicyclic heterocycle or cyclic moiety,preferably, when n is zero, a reduced isoquinoline, and is preferably1,2,3,4,tetrahydroisoquinoline;

in all instances, unless specified, the carbon chains, which may bestraight or branched, contain from 1 to about 12 carbons preferably 1 to6, and most preferably 4-6 carbons, and the cyclic moieties preferablycontain one ring or two fused rings with from 3 to 16 atoms, preferably4 to 12, with 4 to 6 in each ring, in the ring structures.

Unless otherwise stated, the α-amino acids of the compounds of formulaeI and II are preferably in their L-configuration. In their preferredconfiguration with reference to a particular compound, R₁ is S, R₃ is S,and R₅ is R/S. Also, a compound of these formulae may be in free form,e.g., an amphoteric form, or a salt form, e.g., an acid addition or ananionic salt. A compound may be converted to its salt or base form in anart-known manner, one from another. Preferred salts aretrifluoroacetate, hydrochloride, sodium, potassium or ammonium salts,although the scope of the salts embraced is not limited thereto, thescope being extended to include all of those salts known to be used inthe art of chemistry.

Compounds are also provided herein. These compounds may be used in themethods. The compounds have formulae I or II as defined above, but withthe proviso that, when the compounds have formula (I): (1) at least oneof the amino acid residues in the resulting tri-peptide is anon-naturally-occurring α-amino acid or at least one of the R₁, R₃ andR₅ is not a side chain of a naturally-ocurring amino acid; and (2) whenX is an aldehyde, the non-naturally occurring amino acid (or side chainthereof) is other than norleucine or norvaline, and when the compoundshave formula (II) and X is an aldehyde, R₁ cannot be the side chain ofnorleucine or norvaline.

In some embodiments the compounds of formula (II) are also selected suchthat: (1) at least one of the amino acid residues in the resultingdi-peptide is a non-naturally-occurring α-amino acid or at least one ofthe R₁ and R₃ is not a side chain of a naturally-ocurring amino acid;and (2) when X is an aldehyde, the non-naturally occurring amino acid(or side chain thereof) is other than norleucine or norvaline.

In certain preferred embodiments, the compounds have formulae I or II,particularly formula I, as defined above, but with the proviso that: (1)at least one of the amino acid residues in the resulting di ortri-peptide is a non-naturally-occurring α-amino acid or at least one ofthe R₁, R₃ and R₅ is not a side chain of a naturally-ocurring aminoacid; and (2) when R₁ is the side chain from a non-naturally occuringamino acid, it is not the side chain of norleucine or norvaline.

In other preferred embodiments, the compounds have formulae I or II,particularly when the compounds have formula I, as defined above, butwith the proviso that: (1) at least one of the amino acid residues inthe resulting di or tri-peptide is a non-naturally-occurring α-aminoacid or at least one of the R₁, R₃ and R₅ is not a side chain of anaturally-ocurring amino acid; and (2) none of R₁, R₃ and R₅ is the sidechain of norleucine or norvaline.

The compounds provided herein are preferred for use in the methods,particularly the methods of treatment of cognitive disorders.

Pharmaceutical compositions containing a compound of formulae (I) and(II) are provided. In particular, pharmaceutical compositions formulatedfor single dosage administration are provided.

Methods of treatment of diseases, particularly cognitive disorders areprovided and are effected by administering an effective amount of thepharmaceutical compositions. In particular, methods of treating apatient suffering from a neurodegenerative disease selected from amongAlzheimer's disease, cognition deficits, Down's Syndrome, Parkinson'sdisease, cerebral hemorrhage with amyloidosis, dementia pugilistica,head trauma and any disorder characterized by an accumlation of plaquesin the brain, by administering to the patient a therapeuticallyeffective amount of a compound of formulae (I) and (II) or compounds offormulae (I) and (II) in which R₁, R₃, R₅ and R_(B) can all be sidechains of naturally-occurring amino acids are provided.

Methods of treating a patient suffering from a disease statecharacterized by the degeneration of the cytoskeleton arising from athrombolytic or hemorrhagic stroke by administering a therapeuticallyeffective amount of a compound of formulae (I) and (II) are provided.

Assays for detecting compounds that modulate the processing of amyloidprecursor protein (APP) and other related proteins are also provided. Inparticular, assays that detect a relative decrease in the amount ofamyloidogenic Aβ peptide produced by cultured cells that express APP,such as cultured human glioblastoma cell lines that have beentransfected with DNA encoding either a wild-type 695 amino acid isoformof APP or a mutein of APP are provided. A positive in vitro assay occurswhen: (1) there is a decrease in the ˜4-kDa amyloid β-protein (Aβ) inthe medium relative to control cultures; and/or (2) the relative amountof soluble APP (referred to as sAPP, also sβPP and total soluble APP) inthe medium increases; and/or (3) there is a decrease in the amount ofC-terminal fragments of APP larger than 9 kDa in the cell lysate as aresult of differential processing; and/or (4) there is an increase inthe amount of α-sAPP in the medium relative to control cultures.

Methods of detecting markers indicative of neurodegenerative disorderscharacterized by deposition of cerebral amyloid by detecting a decreasein the ratio of α-sAPP to total sAPP or a decrease in the amount ofα-sAPP in a sample of CSF compared to such ratio or amount in controlCSF from individuals who do not have this disorder or compared topredetermined standard ratios and amounts, are also provided herein.

Methods of identifying compounds that are effective for treatingpatients with neurodegenerative disorders characterized by deposition ofcerebral amyloid by administering the compound to a subject anddetecting an increase in the ratio of α-sAPP to total sAPP or anincrease in the amount of α-sAPP in a sample of CSF from the subjectcompared to such ratio or amount in a sample of CSF prior toadministering the compound are also provided herein.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsreferred to herein are incorporated by reference.

As used herein, the term "alkyl" includes the straight, branched-chainand cyclic manifestations thereof, the number of carbon atoms isgenerally specified. Where not specified the alkyl groups preferablycontain from about 1 up to about 10 or 12, more preferably 1 to 6 or 7,and most preferably 4 to 6 carbons. Exemplary of such moieties aremethyl, ethyl, propyl, cyclopropyl, isopropyl, n-butyl, t-butyl,sec-butyl, cyclobutyl, pentyl, cyclopentyl, n-hexyl, n-nonal, n-decyl,cyclohexyl, cyclohexylmethyl, cyclohexylethyl, and the like. Lower alkylrefers to alkyl groups containing six or fewer carbon atoms.

As used herein, heteroatoms are selected from O, N or S.

The term "aryl" within the definitions of X, R_(B), R₁, R₃, R₅, and R₇includes carbocyclic and heterocyclic moieties. Preferred aralkyl andaryl moieties are phenyl, benzyl, phenethyl, 1- and 2-naphthalmethyl, 1-and 2-naphthyl, 2-, 3-, 4-pyridyl, 2- and 3-furyl, 1- and 2-indenyl, 1-and 2-thiophenyl, imidazolyl, indolyl, 2- and 3-thienyl, indole-3-ethyland the residue of 1,2,3,4,tetrahydroisoquinoline. Other carbocycles aresuch fused moieties as pentalenyl, indenyl, naphthaleneyl,naphthylmethyl, azulenyl, heptalenyl, acenaphthylenyl, 9-fluorenyl,phenalenyl, phenanthrenyl, anthracenyl, triphenylenyl, pyrenyl,chryrsenyl, and naphthacenyl. Exemplary of alkynyl is propynyl.Exemplary of alkenyl moieties are 2-methyl-2-propenyl,2-methyl-1-propenyl, propenyl, 1-butenyl, 2-butenyl, 3-butenyl,2,2-difluorophenyl, as well as those straight and branched chainedmoieties having up to two double bonds. Cyclic carbon moietiespreferably contain one or two fused rings typically from 3 up to about16, preferably 4 up to about 12 carbons.

Haloalkyl embraces such moieties as CF₃, --CF₂ H, --CFH₂, CH₂ Cl and CH₂Br and other halo substituted lower alkyls. Exemplary of aryloxyalkenyland aryloxyalkynyl moieties of R_(A) are phenoxymethyl, CF₃ -substitutedphenoxymethyl, benzyloxymethyl, phenoxybutyr-2-ene, 1-phenyl-1-propane,CF₃ -phenoxybutyr-2-ene, CF₃ -benzyloxymethyl, these moieties arepreferred when R_(A) is other than R₁.

In those instances in which a substituent, such as the R₁, R₃, and/or R₅moiety, embrace the residue- or side chain- of a naturally occurringα-amino acid, it is to be noted that each α-amino acid has acharacteristic "R-group," the R-group being the residue- or side chain-attached to the α-carbon atom of the amino acid. For example, theresidue of glycine is H, for alanine it is methyl, for valine it isisopropyl. The specific residues of the naturally occurring α-aminoacids are well known to those of skill in this art see, e.g., A. L.Lehninger, Biochemistry: The Molecular Basis of Cell Structure andFunction, 1970 (or any edition thereafter), Worth Publishers, New York,see, particularly Chapter 4).

As used herein, the residues of naturally occurring α-amino acids arethe residues of those 20 α-amino acids found in nature which areincorporated into protein by the specific recognition of the chargedtRNA molecule with its cognate mRNA codon in humans.

As used herein, the moieties in the peptide analogs provided herein aredesignated according to the system of nomenclature in which the bindingregion of a proteinase is considered as a series of subsites, S, alongthe surface of the enzyme see, Schecter and Berger (Biochem. Biophys.Res. Comm., 27, 157-162 (1967)!. Each subsite binds an individualpeptide residue, P. This system of nomenclature originally designed forpapain, has been adapted to other proteases. Thus, for convenience andin keeping with the customary peptide designations, the moiety bearingthe R₁ side chain (or residue) is designated as the P₁ moiety, themoiety bearing the R₃ side chain (or residue) is designated as the P₂moiety, and that which bears the R₅ moiety is designated as the P₃moiety.

The N-terminal capping moieties represented by the R₇ --(Q)_(n) -- and(R_(B))--CH(R_(A))--(Q)_(n) -- include those moieties that protectmolecules from degradation by aminopeptidases and include, but are notlimited to, such generic groupings as arylcarbonyl, alkylcarbonyl,alkoxycarbonyl, aryloxycarbonyl, aralkyloxycarbonyl, aralkylsulfonyl,alkylsulfonyl, arylsulfonyl, and other equivalently functioning groupsknown in the art.

As defined particularly for the capping groups herein, eitherindividually or as a part of a larger group, "alkyl" means a linear,cyclic, or branched-chain aliphatic moiety of 1 to 20 carbon atoms;"aryl" means an aromatic moiety, e.g., phenyl, of 6 to 18 carbon atoms,unsubstituted or substituted with one or more alkyl, substituted alkyl,nitro, alkoxy, or halo groups; "substituted alkyl" means an alkyl grouphaving a substituent containing a heteroatom or heteroatoms such as N,O, or S; "halo" means Cl or Br; and "alkaryl" means an aryl moiety of 6to 19 carbon atoms having an aliphatic substituent, and, optionally,other substituents such as one or more alkyl, substituted alkyl, alkoxyor amino groups.

Examples of suitable N-terminal blocking groups include, but are notlimited to, formyl, t-butyloxycarbonyl, isopropyloxycarbonyl,allyloxycarbonyl, acetyl, trifluoracetyl, methyl, ethyl, benzyl,benzoyl, acetoacetyl, chloroacetyl, succinyl, phthaloxy,phenoxycarbonyl, methoxysuccinyl, p-methoxybenzenesulfonyl,p-tetuenesulfonyl, isovaleroyl, methanesulfonyl, benzyloxycarbonyl,substituted benzyloxycarbonyl, adipyl, suberyl, phthalamido-,morpholino-, azelayl, dansyl, tosyl, 2,4-dinitrophenyl,fluorenylmethoxycarbonyl, methoxyazelayl, methoxyadipyl, methoxysuberyl,1-adamantanesulfonyl, 1-adamantaneacetyl, 2-carbobenzoyl, phenylacetyl,t-butylacetyl, bis (1-methyl)methyl!acetyl, and thioproline.

As used herein, an effective amount of a compound for treating adisorder is an amount that is sufficient to ameliorate, or in somemanner reduce a symptom or stop or reverse progression of a condition.Such amount may be administered as a single dosage or may beadministered according to a regimen, whereby it is effective.

As used herein, treatment means any manner in which the symptoms orpathology of a condition, disorder or disease are ameliorated orotherwise beneficially altered. Treatment also encompasses anypharmaceutical use of the compositions herein.

As used herein, amelioration of the symptoms of a particular disorder byadministration of a particular pharmaceutical composition refers to anylessening, whether permanent or temporary, lasting or transient that canbe attributed to or associated with administration of the composition.

As used herein, substantially pure means sufficiently homogeneous toappear free of readily detectable impurities as determined by standardmethods of analysis, such as thin layer chromatography TLC!, gelelectrophoresis and high performance liquid chromatography HPLC!, usedby those of skill in the art to assess such purity, or sufficiently puresuch that further purification would not detectably alter the physicaland chemical properties, such as enzymetic and biological activities, ofthe substance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound may, however, be amixture of stereoisomers. In such instances, further purification mightincrease the specific activity of the compound.

As used herein, biological activity refers to the in vivo activities ofa compound or physiological responses that result upon in vivoadministration of a compound, composition or other mixture. Biologicalactivity, thus, encompasses therapeutic effects and pharmaceuticalactivity of such compounds, compositions and mixtures.

As used herein, pharmaceutical activity refers to the activity of thecompounds herein to treat a disorder.

As used herein, the IC₅₀ refers to an amount, concentration or dosage ofa particular compound that achieves a 50% inhibition of a maximalresponse.

As used herein, EC₅₀ refers to a dosage, concentration or amount of aparticular test compound that elicits a dose-dependent response at 50%of maximal expression of a particular response that is induced, provokedor potentiated by the particular test compound.

As used herein, a prodrug is a compound that, upon in vivoadministration, is metabolized or otherwise converted to thebiologically, pharmaceutically or therapeutically active form of thecompound. To produce a prodrug, the pharmaceutically active compound ismodified such that the active compound will be regenerated by metabolicprocesses. The prodrug may be designed to alter the metabolic stabilityor the transport characteristics of a drug, to mask side effects ortoxicity, to improve the flavor of a drug or to alter othercharacteristics or properties of a drug. By virtue of knowledge ofpharmacodynamic processes and drug metabolism in vivo, once apharmaceutically active compound is identified, those of skill in thepharmaceutical art generally can design prodrugs of the compound see,e.g., Nogrady (1985) Medicinal Chemistry A Biochemical Approach, OxfordUniversity Press, New York, pages 388-392!.

As used herein, amyloid precursor protein (APP) is the progenitor ofdeposited amyloidogenic Aβ peptides (Aβ) found in the senile plaques inpatients with diseases, such as Alzheimer's disease (AD), that arecharacterized by such deposition. α-sAPP is an alternative cleavageproduct of APP; its formation precludes formation of Aβ.

As used herein, Cha is cyclohexylalanine.

As used herein, the abbreviations for any substituent groups, protectivegroups, amino acids and other compounds, are, unless indicatedotherwise, in accord with their common usage, recognized abbreviations,or the IUPAC-IUB Commission on Biochemical Nomenclature see, (1972)Biochem. 11:1726!.

A. The tri- and dipeptide analogs

Compounds of formulae (I) and (II): ##STR3## or the hydrates andisosteres, diastereomeric isomers and mixtures thereof, orpharmaceutically acceptable salts thereof, are provided but with theproviso that, when the compounds have formula (I): (1) at least one ofthe amino acid residues in the resulting tri-peptide is anon-naturally-occurring α-amino acid or at least one of the R₁, R₃ andR₅ is not a side chain of a naturally-ocurring amino acid; and (2) whenX is an aldehyde, the non-naturally occurring amino acid (or side chainthereof) is other than norleucine or norvaline, and when the compoundshave formula (II) and X is an aldehyde, R₁ cannot be norleucine ornorvaline.

In some embodiments the compounds of formula (II) are also selected suchthat: (1) at least of one the amino acid residues in the resultingdi-peptide is a non-naturally-occurring α-amino acid or at least one ofthe R₁ and R₃ is not a side chain of a naturally-ocurring amino acid;and (2) when X is an aldehyde, the non-naturally occurring amino acid(or side chain therof) is other than norleucine or norvaline.

In certain preferred embodiments, the compounds have formulae (I) or(II), particularly formula (I), as defined above, but with the provisothat: (1) at least one of the amino acid residues in the resulting di ortri-peptide is a non-naturally-occurring α-amino acid or at least one ofthe R₁, R₃ and R₅ is not a side chain of a naturally-ocurring aminoacid; and (2) when R₁ is the side chain from a non-naturally occuringamino acid, it is not the side chain of norleucine or norvaline.

In other preferred embodiments, the compounds have formulae (I) or (II),particularly formula (I), as defined above, but with the proviso that:(1) at least one of the amino acid residues in the resulting di ortri-peptide is a non-naturally-occurring α-amino acid or at least one ofthe R₁, R₃ and R₅ is not a side chain of a naturally-ocurring aminoacid; and (2) none of R₁, R₃ and R₅ is the side chain of norleucine ornorvaline.

R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R_(A), R_(B), X, Q and n are selectedfrom among (i), (ii), (iii), (iv), (v), (vi), (vii) or (viii) asdescribed above.

Preferred among these compounds, defined with any of the provisos, arethose in which:

R₁ is preferably a straight or branched chain carbon chain containing 2to 6 carbons and one unsaturated, preferably a double bond, or is acyclic moiety containing from 5 to 6 members, and is more preferably2-methyl propene, 2-butene, cyclohexyl, lower alkyl-substitutedcyclohexyl or cyclohexylmethyl;

R₂, R₄, and R₈ are each independently selected from among H or C₁₋₄alkyl, and more preferably methyl or ethyl;

R₃ is C₁₋₄ alkyl, phenyl, benzyl, naphthyl, hydroxyphenyl, 1-aminobutyl,acetamide, and more preferably iso-butyl, benzyl or phenyl;

R₅ is C₁₋₄ alkyl, and more preferably iso-butyl;

R₅ is H or C₁₋₄ alkyl, and more preferably H or methyl; R₇ --(Q)_(n) isacyl, benzyloxycarbonyl (Cbz), 9-fluorenylmethylcarbonate (Fmoc), Ac,Boc, tosyl, with Cbz, Ac and Fmoc being more preferred, and Cbz and Acmost preferred;

Q is --C(O)--, --S(O)₂ -- and --O--C(O), with --C(O)-- and --O--C(O)being more preferred, and --O--C(O) most preferred;

R_(B) is C₁₋₄ alkyl or C₂₋₄ alkenyl, and more preferbly iso-butyl;

R_(A) =--(T)_(m) --(D)_(m) --R₁, in which T is oxygen, or nitrogen, withoxygen being more preferred, and D is C₁₋₄ alkyl or C₂₋₄ alkenyl, with amono-unsaturated C₃₋₄ alkenyl being more preferred; and

X is aldehyde, α-ketoester, α-ketoamide, trifluoromethylketone,diazomethylketone, or nitrile, with an aldehyde, α-ketoester orα-ketoamide being more preferred.

Also among preferred compounds are those of formula (II) in which R_(B)and R_(A) and the atom to which each is attached and (Q)_(n) form(2SR)-N- (2S)-2-benzoxy-4-methylpentanoyl! or (2SR)-N- (2R)-2-(1'-phenyl-1'-propene)-4-methylpentanoyl or valeroyl.

When R₁, R₃, and R₅ are a side-chain from other than a residue of anaturally occurring α-amino acid, it is preferred that such moiety is astraight or branched carbon chain, preferably containing at least oneunsaturated bond, preferably a double bond, and 2 to 10, preferably 4 to7, more preferably 4-6 carbon atoms in the chain, such as, but notlimited to, 2-methyl propene and 2-butone, or is a cylic moiety,preferably containing 4-6 members, more preferably is cyclohexylmethyl.The resulting residues including such moieties include, but are notlimited to, 2-amino-4-methyl-4-pentenoic acid, 2-amino-4-hexenoic acid,cyclohexylalanine and cyclohexylglycine, with(2S)-2-amino-4-methyl-4-pentenoic acid and (2S)-2-amino-4-hexenoic acidbeing preferred.

When the compounds are used in the methods of treating neurodegenerativediseases and cognitive disorders provided herein, the side chains fromnorvaline and leucine are also preferred.

In particular, preferred compounds are those in which at least one ofR₁, R₃, and R₅ is 2-methyl-propene, 2-butene, cyclohexyl orcyclohexylmethyl. More preferred are those in which R₁, R₃, and R₅ are2-methyl-propene, 2-butone, cyclohexyl or cyclohexylmethyl, and X isC(O)H, C(O)--C(O)OR_(D), C(O)--C(O)--NR_(D) R_(D), C(O)CHN₂, C(O)CF₃, orC.tbd.N.

Preferred heterocyclic ring moieties containing R₁ and R₂ and the atomsto which they are at attached, when R₈ is H, are morpholino,thiomorpholino, pyrrolidinyl, or V-substituted pyrrolidinyl,particularly 4-hydroxy pyrrolidinyl.

Preferred heterocyclic ring moieties containing R₆ and R₇ and the atomsto which they are attached when (Q)_(n) is a carbonyl group are selectedfrom among succinimide, phthalimide or maleimide, with phthalimide beingmore preferred.

Preferred heterocyclic ring moieties containing R₆ and R₇ and the atomsto which they are attached when n in (Q)_(n) is zero are morpholino,thiomorpholino, pyrrolidinyl, V-substituted pyrrolidinyl, particularly4-hydroxy pyrrolidinyl, or 1,2,3,4-tetrahydroisoquinoline.

Preferred moieties, when n is zero, and when R₃ and R₄ or R₅ and R₇,taken together with the atoms to which they are attached, formheterocyclic moieties are morpholino, thiomorpholino, pyrrolidinyl, orV-substituted pyrrolidinyl, particularly 4-hydroxy pyrrolidinyl.

The following are among the preferred compounds provided herein:V-Ac-L-Leu-L-Leu-DL-cyclohexylalaninal; (2SR)-N-Cbz-L-Leu-L-Leu N-2-(4-methyl-4-pentenal)!amide; (2SR)-H-L-Leu N- 2-(ethyl4-methyl-4-pentenoate)!amide hydrochloride; (2SR)-N-(2S)-2-benzoxy-4-methylpentanoyl!-L-Leu N-2-(4-methyl-4-pentenal)!amide; (2SR)-N- (2R)-2-(1'-phenyl-1'-propene)-4-methylpentanoyl!!-L-Leu-N-2-(4-methyl-4-pentenal)!amide; N-Cbz-L-Leu-L-Leu-DL-cyclohexylglycinal;(2SR)-N-Ac-L-Leu-L-Leu N 2-(trans-4-hexenal)!amide;(2SR)-N-Ac-L-Leu-L-Leu N- 2-(4-methyl-4-pentenal)!amide;(2SR)-N-Cbz-L-Leu-L-Phe-DL-cyclohexylalaninal; and(2SR)-N-Cbz-L-Leu-L-Phe-N- 2-(4-methyl-4-pentenal)!amide.

The following are among the preferred compounds for use in the methodsherein: N-Cbz-L-Leu-L-Leu-DL-(methyl)norleucinal;N-Ac-L-Leu-L-Leu-DL-cyclohexylalaninal; (2S)-N-Ac-L-Leu-L-Leu N-2-(pentanenitrile)!-amide; N-Cbz-L-Leu-L-Leu-DL-Nle-CO₂ Et;N-Cbz-L-Leu-L-Leu-DL-Nle-CO₂ H; N-Cbz-L-Leu-L-Leu-DL-Nle-CONHEt;(2SR)-N-Cbz-L-Leu-L-Leu N- 2-(4-methyl-4-pentenal)!amide;(2S)-N-Cbz-L-Leu-L-LeuN- 2-(thiazole-oxo-pentyl!amide;N-Ac-L-Leu-L-Leu-L-Nle-COCHN₂ ; (3SR)-N-Ac-L-Leu-L-Leu N-3-(1,1,1-trifluoro-2-oxo-heptyl)!amide; (2SR)-H-L-Leu N- 2-(ethyl4-methyl-4-pentenoate)!amide hydrochloride; (2SR)-N-(2S)-2-benzoxy-4-methylpentanoyl!-L-Leu N-2-(4-methyl-4-pentenal)!amide; (2SR)-(3S)-N-Cbz-L-Leu-Leu N-3-(2-hydroxy-heptanoic acid)!amide; (2SR)-(3S)-N-Cbz-L-Leu-L-Leu N-3-(methyl 2-hydroxy-heptanoate)!amide; (2SR)-(3S)-N-Cbz-L-Leu-L-Leu N-3-(benzyl 2-hydroxy-heptamide)!amide; (3SR)-(4S)-N-Cbz-L-Leu-L-Leu N-4-(benzyl 3-hydroxy-octamide)!amide; (3S)-N-Cbz-L-Leu-L-Leu N-3-(1-furfylthio-2-oxo-heptane!amide; (2SR)-N- (2R)-2-(1'-phenyl-1'-propene)-4-methylpentanoyl!!-L-Leu-N-2-(4-methyl-4-pentenal)!amide; N-Cbz-L-Pro-L-Leu-L-norleucinal;N-Cbz-L-Leu-L-Leu-DL-cyclohexylglycinal;N-Fmoc-Leu-L-Leu-DL-norleucinal; (2SR)-N-Ac-L-Leu-L-LeuN2-(trans-4-hexenal)!amide; N-Ac-L-Leu-L-Phe-DL-norleucinal;N-Cbz-L-Leu-L-Leu-L-norleucinal; (2SR)-N-Ac-L-Leu-L-Leu N-2-(4-methyl-4-pentenal)!amide; N-Cbz-L-Leu-L-(methyl)Leu-DL-norleucinal;N-Dansyl-L-Leu-L-Leu-DL-norleucinal; N-Ac-L-Phe-L-Leu-DL-norleucinal;(2SR)-N-Cbz-L-Leu-L-Phe-DL-cyclohexylalaninal; and(2SR)-N-Cbz-L-Leu-L-Phe-N- 2-(4-methyl-4-pentenal)!amide.

Also provided herein are the α-ketoesters and α-ketoamides of any of theabove listed compounds. Further intended for use herein are any of theabove-listed compounds in which R₂ and/or R₄ and/or R₆ are methyl (i.e.,the N-methyl derivatives of the compounds).

B Synthesis of the tri- and dipeptide analogs

1. Reaction schemes

The following reaction schemes are depicted to illustrate theconstruction of the peptides provided herein and to illustrate thevariety of reactions that may be used to prepare the intermediates fromwhich compounds of formulae I and II may be prepared. ##STR4##

Alternative methods for preparing aldehydes are depicted in ReactionSchemes A, B and C. In Scheme A, the process is initiated by couplingthe protected amino acid (1) with the N-methyl-N-methoxyaminehydrochloride (2) utilizing EDC(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as its HCl salt) in thepresence of hydroxybenzyl triazole (HOBt) and triethylamine (Et₃ N); thereaction is conducted in CH₂ Cl₂ at room temperature under anhydrousconditions. Upon completion of the coupling reaction, theamine-protecting group is removed by reaction with 4N HCl in dioxane toyield compound (3). The resulting N-methyl-N-methoxyamide derivatives(sometimes referred to as a Weinreb amide) are coupled to a N-protecteddipeptide (4) to produce the analogous N-methyl-N-methoxyamideintermediates (5) which are reduced to the desired aldehydes usingstandard reduction conditions, e.g., by the use of lithium aluminumhydride (LiAlH₄) in tetrahydrofuran (THF) at about 0° C. in an inertatmosphere (Ar or N₂) under anhydrous conditions. After the reaction iscomplete, about 30 min, the reaction is quenched by the addition of, forexample, 10% potassium hydrogen sulfate and then water.

In Reaction Scheme B, the intermediates (A-4; compound 4 prepared inScheme A) are coupled with an ester (derived by a standard Fischeresterification of (1)) and the resulting esters (B-3) are reduced totheir corresponding alcohols (B-4) using lithium borehydride in THF attemperatures of about 0° C. to room temperature; the reduction, ofcourse, being conducted under anhydrous conditions in an inertatmosphere. The resulting alcohols (B-4) are oxidized to the desiredaldehydes using the well-known Swern oxidation procedure.

In Scheme C, the esters (1) are subjected to mild hydrolysis using LiOHand hydrogen peroxide in a methanol-water solvent according to knownconditions. Using the same coupling procedure as described in Scheme A1,the resulting amino acids are coupled with propane sulfide to producethe thioesters (2) which are reduced to their corresponding aldehyde byusing Pd on carbon with triethylsilane under anhydrous conditions atroom temperature under an inert atmosphere.

Reaction Scheme D illustrates the preparation of precursor reactantswhich are amendable to substitutions on the α-carbon atom; thesesubstituents not necessarily being residues of naturally occurringα-amino acids. In this scheme, N-(diphenylmethylene)glycine ethyl ester(1) is treated with lithium bis(trimethylsilyl)amide wherein thereaction is effected in THF under an inert atmosphere at temperatures ofabout -78° C. and the in situ generated base is reacted with theappropriate alkyl halide to effect a nucleophilic displacement. Theso-alkylated intermediates (2) and (4) are subjected to hydrolysis toproduce the amines (3) and (5) which are available for appropriate usein the construction of the desired dipeptides and tripeptides whereinR₁, R₃ or R₅ are side chains other than that of a naturally occurringα-amino acid. Similar such process may be useful in preparing compoundsin which R₂, R₄, or R₆ is an alkylated product.

Reaction Scheme E illustrates the preparation of the P₁ moiety wherein Xis nitrile. The synthesis is initiated by reacting an N-protected aminoacid with iso-butyl chloroformate in the presence of 4-methylmorpholineat -78° C. under an inert atmosphere and the resulting mixed anhydridederivatives are treated with ammonia gas at -78° C. to form thecorresponding amides (2). The amide is converted to its nitrile bydehydration with tosyl chloride in pyridine followed by the removal ofthe Boc protecting group by hydrolysis with 4N HCl in dioxane. Followingtheir preparation, the P₁ moieties are coupled with the appropriate P₂P₃ moiety (A-4), or with the appropriate P₂ moiety (J-5) to produce thenitriles of E-5 or in the case of coupling with the P₂ moiety of J-5,the corresponding nitrile analogues to the aldehydes (J-7)!.

Reaction Scheme F illustrates the preparation of compounds of a P₁moiety wherein X is a C(O)W, defined as above, C₁₋₆ alkyl or aralkyl. Ineffecting the preparations, the N-methyl-N-methoxyamide derivative (1)is treated with a lithiothiazole nucleophile generated in situ toproduce a ketothiazole which is deprotected by hydrolysis with 4N HCl indioxane to obtain compounds (2) which are then coupled to theappropriate P₂ P₃ moieties to obtain the derivatives of compound (3) orwith the appropriate P₂ moieties (i.e., (R_(A))CH(R_(B))--C(O)N(R₄)--CH₂(R₃)C(O)OH) to obtain the desired dipeptides. Of course, the lithioderivative of thiazole may be replaced with litho derivatives of otheraryl, aralkyl and alkyl moieties and by following substantially the sameprocedures, the corresponding di- and tri- peptides may be obtained.

Reaction Scheme G illustrates the preparation of compounds of formulae Iand II wherein the X moiety is a haloketone. The reaction is initiatedby reacting an R₁ -substituted nitromethane with a trifluoromethylacetal (2) in DMF in the presence of potassium carbonate at about 60° C.to yield a 1,1,1-trifluoro-2-hydroxy-3-nitro derivative (3) which arereduced with H₂ in the presence of Raney Nickel to yield thecorresponding amines (4). By appropriate coupling, the alcohols of (6)may be produced which would then be subjected to Dess-Martin oxidationto produce the desired CF₃ analogues (7). By use of mono- anddi-fluoromethyl analogues of formula (2) and by following substantiallythe same procedures, there are produced the corresponding --CH₂ F and--CHF₂ ketone analogues.

Reaction Scheme H illustrates the preparation of compounds of formulae Iand II wherein the X represents an α-ketoester or α-ketoamide(C(O)C(O)OR_(D) or C(O)C(O)NR_(D) R_(D), respectively). In this process,the peptides are subjected to a modified Dakin West reaction to generatean enol ester which is subjected to a basic hydrolysis to obtain theα-keto esters (2). To transform the α-keto esters to their correspondingamides, the ketone is protected by a ketal formation by reaction withHSCH₂ CH₂ SH in the presence of a BF₃ etherate. Treatment with ethanolat 0° C. in the presence of the appropriate amines forms the amidemoiety and deprotects the ketal to form the α-keto amide (4).Alternatively, hydrolysis of intermediate (2) produces the α-keto acid(5).

Reaction Scheme I illustrates the preparation of compounds of formulae Iand II wherein the X moiety is a diazomethane which may be converted toa halomethyl ketone. In this process the amine protected peptides aresubjected to reaction with iso-butyl chloroformate in the presence of4-methylmorpholine in CH₂ Cl₂ at -78° C. The mixed anhydride derivativesare reacted with diazomethane according to standard procedures wellknown in the art. If desired, the diazoketones of formulae II may betreated with the appropriate acid (e.g., HF, HCl, HBr), in pyridine toafford the desired ketohalomethyl derivatives.

Reaction Scheme J illustrates the preparation of compounds of formulae Iand II wherein the amino terminus of the peptide is modified to producecompounds embraced within the scope of the R_(A) (R_(B))--CH--(Q)_(n)--! of formulae II as well as compounds which fall within the scope ofR₇ --(Q)_(n) -- of fomulae I wherein (Q)_(n) is C(O). The scheme isinitiated by the conversion of the acid (1) to its acid chloride.Treatment of the acid chloride with the appropriate chiral auxiliary(e.g., 4S,5R-(-)-4-methyl-5-phenyl-oxazolidinone) in the presence oftriethylamine in CH₂ Cl₂ produces the imides (2). These imides aretreated with lithium bis(trimethylsilyl)amide in tetrahydrofuran at -78°C. (initially) and the resulting activated moiety, generated in situ, issubjected to a stereoselective alkylation using a haloelectrophile. Thealkylation is completed as the mixture warms to room temperature toproduce compounds (3) as pure enantiomers. Hydrolysis with lithiumhydroxide in hydrogen peroxide produces the acids (4) which are coupledto an amino acid (in its ester form) using the described EDC, HOBt, Et₃N coupling process, followed by hydrolysis of the ester. The hydrolyzedamino acid is subjected to another coupling reaction with an amino acid(in its ester form), and the ester of the resulting dipeptide is reducedwith lithium borohydride to its corresponding alcohol (6). The alcoholis converted to its aldehyde using the Swern oxidation procedure. Ofcourse, by selecting the appropriate chiral auxiliary and substantiallyfollowing the outlined procedure, other stereospecific enantiomers maybe produced.

Reaction Scheme K illustrates the formation of a dipeptide wherein R_(B)may represent an aryloxy, aralkoxy or an alkoxy in an enantiomeric pureisomer. The reaction is initiated by a two step process wherein (a)compound (1) is hydrodeaminated by treatment with NaNO₂ in HCl and (b)an esterification of the acid with an alkyl halide in the presence ofDMF and cesium carbonate to produce compounds (2). These are treatedwith a 2,2,2-trichloroacetimate derivatives in the presence oftrifluoromethanesulfonic acid in CH₂ Cl₂ to obtain the desired ester andthe ester moiety is hydrolyzed with lithium hydroxide in peroxide and amethanol-water solvent to produce the enantiomers of formula (3). Theisomers are coupled with the appropriate P₂ P₁ moieties (as esters), theresulting esters (5) are reduced to their corresponding alcohols whichare then oxidized to the corresponding aldehydes (6).

Reaction Scheme L illustrates the preparation of compounds of formulae Iand II wherein the X is defined by the moiety C(O)CH₂ Y. The N-protecteddiazoketone derivatives of compound (1) are subjected to an additionreaction with an hydrohalic acid, preferably HCl, in pyridine to producehalomethyl derivatives (2), which are subjected to a nucleophilicdisplacement reaction using an activated anion of the desired Y moiety,(e.g., Y), to afford compounds (3). Standard hydrolysis reactions removethe N-protecting group followed by the usual coupling procedures withthe desired P₂ P₃ moieties (e.g., compounds A-4) to produce the desiredcompounds (5).

Reaction Scheme M illustrates the preparation of compounds of formulae Iand II wherein the X is defined by the moieties (a)--CH(OH)--C(O)--NR_(D) R_(D) and (b) --CH(OH)--C(O)OR_(D). The processconveniently starts with the obtention of the aldehyde (2) by reducingthe N-methyl-N-methoxy amide derivative (1), followed by preparation ofthe cyanohydrin (3) which is hydrolyzed to its free acid (4) usingstandard and well known reaction techniques. Coupling of the desireddipeptides (i.e., the P₂ P₃ moiety) to the acid (4) is effected by theuse of an activated iso-butyl chloroformate in the presence of4-methylmorpholine at -78° C. in an inert atmosphere under anhydrousconditions to afford the acid (5). The acids (5) may be esterified toits corresponding ester or may be coupled with an amine (NR_(D) R_(D))to produce the desired amides (6). Of course, using substantially thesame procedure but replacing the P₂ P₃ moieties of formula A-4 with theappropriate P₂ moiety, analogous dipeptides may be prepared.

Alternatively, compounds (2) may be transformed to their N-protected(preferably a Boc group) alkyl ester by reaction with ethylacetate inthe presence of LDA to produce compounds (8) which are hydrolyzed with4N HCl in dioxane to remove the protecting group to produce thecorresponding β-hydroxy ethyl esters (9). These esters are then coupledwith compounds (A-4) and the resulting compounds are hydrolyzed to theirβ-hydroxy acids or they may be coupled to form their β-hydroxyamides ofcompounds (11).

Reaction Scheme N illustrates the process by which compounds of formulaeI and II wherein the R₂, R₄, or R₆ represent an R_(D) moiety other thanH. In essence, the procedure utilizes standard N-protection,N-alkylation esterfication and de-protection procedures such as thoseexemplified in the depicted schemes. Of course, although the reactionscheme depicts N-alkylation at the projected P₁ moiety, any of the P₂and P₃ moieties may be similarly N-alkylated by appropriate selection ofthe starting materials followed by the coupling procedures required toconstruct the desired peptides of formulae I and II.

Throughout the above presentation of the methods useful for preparingthe compounds herein, particularly as it relates to the foregoingreaction schemes, the full embodiment of the entire scope of thecompounds (as defined in formulae I and II) was not depicted within allof the structures illustrated for each of the reactants andend-products. The state of the art is such that one of skill in the artwould be able to extend these specific illustrations to embrace theimplied generic teachings by the use of analogy reasoning to prepare thedesired compounds embraced within the scope of formulae I and II. Forexample, in Reaction Scheme A it is to be noted that the final compounds(i.e., compounds (6) of that reaction scheme) are tripeptides. It can beseen that by substituting the reactant (4) with the appropriate α-aminoacid, and by following the teachings herein, dipeptides would result.Similarly one of skill in the art could utilize the final product ofReaction Scheme D in preparing any of the compounds of formulae I and IIbearing the R₁ side chain functionality, which is other than a residueof a naturally occurring α-amino acid. Similarly, in Reaction Scheme F,the preparation of a thiazole derivative is achieved by coupling theN-methoxy-N-methylamide derivative of a precursor for preparing adepicted tripeptide (3). A dipeptide bearing a thiazole derivative couldbe prepared by the application of the analogy reasoning possessed by aperson of skill in the art. Similarly, lithio derivative of anotherheterocycle in which X is C(O)aryl and the aryl moiety is other than athiazole embraced within the scope of the compounds herein could beprepared.

Thus, the scope of those compounds preparable by the methods of theforegoing reaction schemes is not limited to the specific compoundsdepicted but rather to those compounds defined by formulae I and IIusing the teachings already available in the art and which areexemplified to illustrate such teachings.

2. Procedures to effect the reaction schemes

The construction of the tri- and dipeptide analogs of Formulae I and IImay be effected using procedures and techniques well known in the artand described herein. Many of the necessary starting materials and thereactants utilized are known and may also be commercially available. Inthose instances in which they are not generally available, they mayreadily be generated by analogous use of known chemical processes andtechniques readily available in the scientific and patent literature oras described herein.

As the reaction schemes depicted herein (Schemes A-N) extensivelyutilize coupling and oxidation procedures, the following elaborates avariety of the procedures that may be functional alternatives to thosespecifically mentioned within the depicted schemes.

As a preferred oxidation procedure, the Swern oxidation is effected byreacting 2 to 10 equivalents of dimethyl sulfoxide (DMSO) with about 1to 6 equivalents of trifluoroacetic anhydride (CF₃ CO)₂)! or oxalylchloride --(COCl)₂ !. The reactants are dissolved in an inert solvent,e.g., methylene chloride (CH₂ Cl₂), the reactor is under an inertatmosphere under anhydrous conditions at temperatures of about -80° C.to -50° C. to form an in situ sulfonium adduct to which is added about 1equivalent of the alcohols (e.g., B-4). Preferably, the alcohols aredissolved in an inert solvent, e.g., CH₂ Cl₂ or minimum amounts of DMSO,and the reaction mixture is allowed to warm to about -50° C. (for about10-20 minutes) and then the reaction is completed by adding 3 to 10equivalents of a tertiary amine, e.g., triethylamine, N-methylmorpholine, etc. Following oxidation, the desired intermediates areisolated and are ready for the next step in the reaction sequence.

A modified Jones oxidation procedure may conveniently be effected byreacting the alcohols with pyridinium dichromate by contacting thereactants in a water-trapping sieve powder, (e.g., a grounded 3 Angstrommolecular sieve) in the presence of glacial acetic acid at about 5° C.to 50° C., preferably at room temperature.

Alternatively, 1 to 5 equivalents of a chromic anhydride-pyridinecomplex i.e., a Sarett reagent prepared in situ (see, e.g., Fieser andFieser "Reagents for Organic Synthesis" Vol. 1, pp. 145 and Sarett, etal., J.A.C.S. 25, 422 (1953))! that is prepared in situ in an inertsolvent (e.g., CH₂ Cl₂) in an inert atmosphere under anhydrousconditions at about 0° C. to 50° C. to which complex is added 1equivalent of the alcohols allowing the reactants to interact for about1 to 15 hours, followed by the isolation of the desired product.

Another alternative process for the converting of alcohols to thedesired ketones is an oxidation reaction that employs periodane i.e.,1,1,1-triacetoxy-1,1-dihydro, 1,2-benzoxidol 3-(1-H)-one (see DessMartin, J. Org. Chem. 48, 4155, (1983))!. This oxidation is effected bycontacting 1 equivalent of the alcohols with 1 to 5 equivalents ofperiodane (preferably 1.5 equivalents) in suspension in an inert solvent(e.g., CH₂ Cl₂) under an inert atmosphere (preferably nitrogen) underanhydrous conditions at about 0° C. to 50° C. (preferably roomtemperature), and allowing the reactants to interact for about 1 to 48hours.

A solid phase sequential coupling procedure can be performed usingestablished methods such as use of an automated peptide synthesizer. Inthis procedure, an amino protected amino acid is bound to a resinsupport at its carboxyl terminus, the protected amine is deprotectedwhere the peptide linkage is desired, the amino group neutralized with abase and the next amino protected amino acid in the desired sequence iscoupled in a peptide linkage. The deprotection, neutralization andcoupling steps are repeated until the desired peptide is synthesized.The compounds provided herein are thus synthesized from their carboxylterminal end to their amino terminal end. The amino protected amino acidcan be a conventional amine acid, a derivative or isomer thereof, or aspacer group. The resin support employed can be any suitable resinconventionally employed in the art for the solid phase preparation ofpolypeptides. The preferred resin is polystyrene which has beencross-linked with from about 0.5 to about 3% divinyl benzene, which hasbeen either benzhydrylamidated, chloromethylated or hydroxymethylated toprovide sites for amide or ester formation with the initially introducedamino protected amino acid.

An example of a hydroxymethyl resin is described by Bodansky et al.Chem. Ind. (London) 38, 1597-98 (1966)!. The preparation of chloromethyland benzhydrylamine resins are described by Stewart et el. "Solid PhasePeptide Synthesis," 2nd Edition, Pierce Chemical Co., Rockford, Ill.(1984). Chapter 2, pp, 54-55!. Many of these resins are availablecommercially. In general, the amino protected amino acid which isdesired on the carboxy-terminal end of the peptide is bound to the resinusing standard procedures and practices as are well known andappreciated in the art. For example, the amino protected amino acid canbe bound to the resin by the procedure of Gisin Helv. Chem. Acta, 56,1476 (1973)!. When it is desired to use a resin containing abenzhydrylamine moiety as the resin binding site an amino protectedamino acid is coupled to the resin through an amide linkage between itα-carboxylic acid and the amino moiety of the resin. The coupling iseffected using standard coupling procedures as described below. Manyresin-bound amino acids are available commercially.

The α-amino protecting group employed with each amino acid introducedinto the polypeptide sequence may be any such protecting group known inthe art. Among the classes of amino protecting groups contemplated are:(1) acyl type protecting groups such as formyl, trifluoroacetyl,phthalyl, p-toluenesulfonyl (tosyl), benzenesulfonyl,nitrophenylsulfonyl, tritylsulfonyl, o-nitrophenoxyacetyl, andα-chlorobutryl; (2) aromatic urethane type protecting groups such asbenzyloxycarbonyl and substituted benzyloxycarbonyls such asp-chlorobenzyioxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, and benzhydryloxycarbonyl;(3) aliphatic urethane protecting groups such as t-butyloxycarbonyl(Boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl,and allyloxycarbonyl; (4) cycloalkyl urethane type protecting groupssuch as cyclopentyloxycarbonyl, adamantyloxycarbonyl, andcyclohexyloxycarbonyl; (5) thiourethane type protecting groups such asphenylthiocarbonyl; (6) alkyl type protecting groups such astriphenylmethyl (trityl) and benzyl (Bn); (7) trialkylsilane protectinggroups such as trimethylsilane, 4--(4-chlorophenyl)sulfonylaminocarbonyl!phenyl carbonyl, and 4--(4-bromophenyl)sulfonylaminocarbonyl!phenyl carbonyl. The preferredα-amino protecting group is t-butyloxycarbonyl (Boc); its use as anα-amino protecting group for amino acids is well known to those of skillin the art ( see, e.g., by Bodansky et al. in "The Practice of PeptideSynthesis," Springer-Verlag, Berlin (1984), p. 20!.

Following the coupling of the amino protected amino acid to the resinsupport, the α-amino protecting group may be removed using any suitableprocedure such as by using trifluoroacetic acid, trifluoroacetic acid inCH₂ Cl₂, or HCl in dioxane. The deprotection is carried out at atemperature of between 0° C. and room temperature. Other standardcleaving reagents may be used for removal of specific amino protectinggroups under conditions well known and appreciated in the art.

After removal and neutralization of the α-amine protecting group, thenext desired amine-protected amino acid is coupled through a peptidelinkage. This deprotection, neutralization and coupling procedure isrepeated until a peptide of the desired sequence is obtained.Alternatively, multiple amino acid groups may be coupled by the solutionmethod prior to coupling with the resin supported amino acid sequence.

The selection and use of an appropriate coupling reagent is within theskill of the skilled artisan. Particularly suitable coupling reagentswhere the amino acid to be added is Gln, Asn, or Arg includeN,N-dicyclohexyl-carbodiimide and 1-hydroxybenzotriazole. The use ofthese reagents prevents nitrile and lactam formation. Other couplingagents are (1) other carbodiimides (e.g.,N-ethyl-N'-(γ-dimethylaminopropylcarbodiimide); (2) ketenimines; (3)isoxazolium salts (e.g., N-ethyl-5-phenylisoxazolium-3-sulfonate); (4)monocyclic nitrogen-containing heterocyclic amides of aromatic charactercontaining one through four nitrogens in the ring such as imidazolides,pyrazolides, and 1,2,4-triazolides (specific heterocyclic amides thatare useful include N,N-carbonyldiimidazole andN,N-carbonyl-di-1,2,4-triazole); (5) alkoxylated acetylene (e.g.,ethoxyacetylene); (6) reagents which form a mixed anhydride with thecarboxyl moiety of the amino acid (e.g., ethyl chloroformate andiso-butyl chloroformate) or the symmetrical anhydride of the amino acidto be coupled (e.g., Boc-Ala-O-Ala-Boc); (7) nitrogen-containingheterocyclic compounds having a hydroxyl group on one ring nitrogen(e.g., N-hydroxyphthalimide, N-hydroxysuccinimide, and1-hydroxybenzetriazole). Other activating reagents and their use inpeptide coupling are described by Kapoor J. Pharm. Sci, 59, 1-27(1970)!. Use of the symmetrical anhydride as the coupling agent is thegenerally preferred amino acid coupling method herein.

The preferred coupling method for Gln, Asn and Arg is to react theprotected amino acid, or derivatives or isomers thereof, withN,N-dicyclohexylcarbodiimide and 1-hydroxybenzotriazole (1:1) inN,N-dicyclohexylcarbodiimide (DMF) in the presence of the resin orresin-bound amino acid or peptide. The preferred coupling method forother amino acids involves reacting the protected amino acid, orderivative or isomer thereof, with N,N-dicyclohexylcarbodiimide in CH₂Cl₂ to form the symmetrical anhydride. The symmetrical anhydride is thenintroduced into the solid phase reactor containing the resin orresin-bound amino acid or peptide, and the coupling is carried out in amedium of DMF, or CH₂ Cl₂, or DMF: CH₂ Cl₂ (1:1). A medium of DMF ispreferred. The success of the coupling reaction at each stage of thesynthesis is monitored by a ninhydrin test as described by Kaiser et al.Analyt. Biochem. 34, 595 (1970)!. In cases where incomplete couplingoccurs, the coupling procedure is repeated. If the coupling is stillincomplete, the deprotected amine is capped with a suitable cappingreagent to prevent its continued synthesis. Suitable capping reagentsand the use thereof are well known and appreciated in the art. Examplesof suitable capping reagents are acetic anhydride and acetylimidazole asdescribed by Stewart et al. "Solid Phase Peptide Synthesis," 2nd Ed.,Pierce Chemical Co., Rockford, Ill. (1984), Chapter 2, p. 73!.

After the desired amino acid sequence has been obtained, the peptide iscleaved from the resin. This can be effected by procedures which arewell known and appreciated in the art, such as by hydrolysis of theester or amide linkage to the resin. It is preferred to cleave thepeptide from the benzhydrylamine resin with a solution of dimethylsulfide, p-cresol, thiocresol, or anisole in anhydrous hydrogenfluoride. The cleavage reaction is preferably carried out attemperatures between about 0° C. and about room temperature, and isallowed to continue preferably from between about 5 minutes to about 5hours.

As is known in the art of solid phase peptide synthesis, many of theamino acids bear side chain functionalities requiring protection duringthe preparation of the peptide. The selection and use of an appropriateprotecting group for these side chain functionalities is within theability of those skilled in the art and will depend upon the amino acidto be protected and the presence of other protected amino acid residuesin the peptide. The selection of such a side chain protection group iscritical in that it must not be removed during the deprotection andcoupling steps of the synthesis. For example, when Boc is used as theα-amino protecting group, the following side chain protecting groups aresuitable: p-toluenesulfonyl (tosyl) moieties can be used to protect theamino side chains of amino acids such as Lys and Arg; p-methylbenzyl,acetamidomethyl, benzyl (Bn), or t-butylsulfonyl moieties can be used toprotect the sulfide-containing side chains of amino acids such ascysteine, homocysteine, penicillamine and the like or derivativesthereof; benzyl or cyclohexyl ester moieties can be used to protectcarboxylic acid side chains of amino acids such as Asp, Glu; a benzylether can be used to protect the hydroxyl-containing side chains ofamino acids such as Ser and Thr; and a 2-bromocarbobenzoxy (2Br-Cbz)moiety can be used to protect the hydroxyl-containing side chains ofamino acids such as Tyr. These side chain protecting groups are addedand removed according to standard practices and procedures well known inthe art. It is preferred to deprotect these side chain protecting groupswith a solution of anisole in anhydrous hydrogen fluoride (1:10).Typically, deprotection of side chain protecting groups is performedafter the peptide chain synthesis is complete but these groups canalternatively be removed at any other appropriate time. It is preferredto deprotect these side chains at the same time as the peptide iscleaved from the resin.

The compounds are then isolated and purified by standard techniques. Thedesired amino acids, derivatives and isomers thereof can be obtainedcommercially or can be synthesized according to standard practices andprocedures well known in the art.

C. Identification of preferred compounds using assays that identifycompounds that modulate processing of amyloid precursor protein (APP)

Compounds provided herein modulate the processing of proteins, such asamyloid precursor protein (APP), involved in neurodegenerative diseases.The ability of compounds to modulate processing of APP can bedemonstrated in a variety of ways. For example, compounds can beevaluated for the ability to modulate generation of Aβ or α-sAPP.

1. In vitro assays

The compounds provided herein yield a positive result in one or more invitro assays that assess the effects of test compounds on processing ofAPP. In particular, in vitro assays systems for identifying suchcompounds are provided herein. These assays evaluate the effects of atest compound on processing of APP and use cultured human glioblastomacell lines that have been transfected with DNA encoding either awild-type 695 amino acid isoform of APP or a mutein of that APP thatcontains changes (in this case two or three amino acid changes have beenmade) that appear to make the molecule more susceptible to proteolyticcleavage that results in increased production of Aβ see, e.g., Mullan etal. (1992) Nature Genet. 1:345-347!.

In performing these assays, a test compound is added to the culturemedium and, after a selected period of time, the culture medium and/orcell lysates are analyzed using immunochemical assays to detect therelative amounts of Aβ, total soluble APP (sAPP), a portion of sAPPdesignated α-sAPP, and C-terminal fragments of APP. In particular, theculture medium and cell lysates are analyzed by immunoblotting coupledwith laser scanning densitometry and ELISAs using several differentantibodies. A positive test occurs when: (1) there is a decrease in the˜4-kDa amyloid β-protein (Aβ) in the medium relative to control cultures(4-kDa assay); and/or (2) the relative amount of total sAPP in themedium increases; and/or (3) there is a decrease in the amount ofC-terminal amyloidogenic fragments larger than 9 kDa and smaller than 22kDa in the cell lysate as a result of differential processing; and/or(4) there is an increase in the amount of α-sAPP in the medium relativeto control cultures. Control cultures can be cultures that have not beencontacted with the compound. The Aβ assay is done using cells (e.g., HGB717/Swed) that have been transfected with DNA encoding the mutein APP;the other assays are performed using cells, such as HGB695 cells, thathave been transfected with DNA encoding a wild-type APP.

Preferred compounds have activity that is at least 2-fold, preferably5-fold, most preferably 10-fold greater activity thanN-Acetylleucylnorleucinal see, e.g., EP 0 504 938 A2; and Sherwood etal. (1993) i Proc. Natl. Acad. Sci. U.S.A. 90:3353-3357! in at leastone, preferably the Aβ assay, of these assays.

2. The amount of α-sAPP and the ratio of α-sAPP to total sAPP incerebrospinal fluid (CSF) as an indicator of Alzheimer's Disease (AD)and the effectiveness of therapeutic intervention

The relative amount of α-sAPP and the ratio of α-sAPP to total sAPP inCSF are shown herein to be useful markers in the detection ofneuro-degenerative disorders characterized by cerebral deposition ofamyloid (e.g., AD) and in monitoring the progression of such disease.Furthermore, assay systems incorporating these markers can be used inmonitoring therapeutic intervention of these diseases.

As shown in EXAMPLE 32, the amount of α-sAPP and the ratio of α-sAPP tototal sAPP in CSF samples can be used as an indicator of Alzheimer'sDisease and other neurodegenerative disorders. For purposes herein, thisamount and/or the ratio can also be used to assess the effectiveness ofcompounds provided herein in treating Alzheimer's Disease andneurodegenerative disorders.

It has been found that patients with suspected Alzheimer's disease (asdiagnosed by other indicia, or confirmed by autopsy) have astatistically significant lower ratio of α-sAPP to total sAPP in CSF andalso have statistically significant lower levels of α-sAPP. Therefore,by comparison with non-Alzheimer's disease controls or by existence of aratio lower than a predetermined standard, based, for example, onaverages in samples from large numbers of unafflicted individuals, or anamount of α-sAPP lower than a predetermined standard, Alzheimer'sdisease or, depending upon other indications, another neurodegenerativedisease is indicated.

Compounds, such as those provided herein, that alter this ratio or thelevel of α-sAPP closer to that of individuals who do not have aneurodegenerative disorder characterized by the cerebral deposition ofamyloid are considered useful for treating these disorders.

3. In vivo assays

The ability of compounds to modulate processing of APP can also beevaluated in vivo see, e.g., Kowall et al. (1991) Proc. Natl. Acad. Sci.U.S.A. 88:7247-7251!. Compounds can be administered through a canulaimplanted in the cranium of a rat or other suitable test animal see,e.g., Lamb et al. (1993) Nature Genet. 5:22-29; Pearson et al. (1993)Proc. Natl. Acad. Sci. U.S.A. 90:10578-10582!. After a predeterminedperiod of administration the rats are sacrificed. The hippocampi areevaluated in immunoblot assays or other suitable assays to determine therelative level of α-sAPP and C-terminal fragments of APP compared tountreated control animals. Compounds that result in relative increasesin the amount of α-sAPP are selected.

D. Formulation of pharmaceutical compositions

Compositions are provided that contain therapeutically effective amountsof the compounds of formulae (I) and (II). The compounds are preferablyformulated into suitable pharmaceutical preparations such as tablets,capsules or elixirs, for oral administration or in sterile solutions orsuspensions for parentoral administration, as well as transdermal patchpreparation. Typically the compounds described above are formulated intopharmaceutical compositions using techniques and procedures well knownin the art.

About 10 to 500 mg of a compound or mixture of compounds for Formulae Iand II or a physiologically acceptable salt is compounded with aphysiologically acceptable vehicle, carrier, excipient, binder,preservative, stabilizer, flavor, etc., in a unit dosage form as calledfor by accepted pharmaceutical practice. The amount of active substancein those compositions or preparations is such that a suitable dosage inthe range indicated is obtained.

To prepare compositions, one or more compounds of formulae (I) and (II)are mixed with a suitable pharmaceutically acceptable carrier. Uponmixing or addition of the compound(s), the resulting mixture may be asolution, suspension, emulsion or the like. Liposomal suspensions mayalso be suitable as pharmaceutically acceptable carriers. These may beprepared according to methods known to those skilled in the art. Theform of the resulting mixture depends upon a number of factors,including the intended mode of administration and the solubility of thecompound in the selected carrier or vehicle. The effective concentrationis sufficient for ameliorating the symptoms of the disease, disorder orcondition treated and may be empirically determined.

Pharmaceutical carriers or vehicles suitable for administration of thecompounds provided herein include any such carriers known to thoseskilled in the art to be suitable for the particular mode ofadministration. In addition, the active materials can also be mixed withother active materials that do not impair the desired action, or withmaterials that supplement the desired action or have other action. Thecompounds may be formulated as the sole pharmaceutically activeingredient in the composition or may be combined with other activeingredients.

In instances in which the compounds exhibit insufficient solubility,methods for solubilizing compounds may be used. Such methods are knownto those of skill in this art, and include, but are not limited to,using cosolvents, such as dimethylsulfoxide (DMSO), using surfactants,such as tween, or dissolution in aqueous sodium bicarbonate. Derivativesof the compounds, such as salts of the compounds or prodrugs of thecompounds may also be used in formulating effective pharmaceuticalcompositions.

The concentrations of the compounds are effective for delivery of anamount, upon administration, that ameliorates the symptoms of thedisorder for which the compounds are administered. Typically, thecompositions are formulated for single dosage administration.

The compounds of formulae (I) and (II) may be prepared with carriersthat protect them against rapid elimination from the body, such as timerelease formulations or coatings. Such carriers include controlledrelease formulations, such as, but not limited to, microencapsulateddelivery systems,

The active compound is included in the pharmaceutically acceptablecarrier in an amount sufficient to exert a therapeutically useful effectin the absence of undesirable side effects on the patient treated. Thetherapeutically effective concentration may be determined empirically bytesting the compounds in known in vitro and in vivo model systems forthe treated disorder.

The compositions can be enclosed in ampules, disposable syringes ormultiple or single dose vials made of glass, plastic or other suitablematerial. Such enclosed compositions can be provided in kits.

The concentration of active compound in the drug composition will dependon absorption, inactivation and excretion rates of the active compound,the dosage schedule, and amount administered as well as other factorsknown to those of skill in the art.

The active ingredient may be administered at once, or may be dividedinto a number of smaller doses to be administered at intervals of time.It is understood that the precise dosage and duration of treatment is afunction of the disease being treated and may be determined empiricallyusing known testing protocols or by extrapolation from in vivo or invitro test data. It is to be noted that concentrations and dosage valuesmay also vary with the severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that theconcentration ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed compositions.

If oral administration is desired, the compound should be provided in acomposition that protects it from the acidic environment of the stomach.For example, the composition can be formulated in an enteric coatingthat maintains its integrity in the stomach and releases the activecompound in the intestine. The composition may also be formulated incombination with an antacid or other such ingredient.

Oral compositions will generally include an inert diluent or an ediblecarrier and may be compressed into tablets or enclosed in gelatincapsules. For the purpose of oral therapeutic administration, the activecompound or compounds can be incorporated with excipients and used inthe form of tablets, capsules or troches. Pharmaceutically compatiblebinding agents and adjuvant materials can be included as part of thecomposition.

The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a binder,such as, but not limited to, gum tragacanth, acacia, corn starch orgelatin; an excipient such as microcrystalline cellulose, starch andlactose, a disintegrating agent such as, but not limited to, alginicacid and corn starch; a lubricant such as, but not limited to, magnesiumstearate; a gildant, such as, but not limited to, colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; and aflavoring agent such as peppermint, methyl salicylate, and fruitflavoring.

When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier such as a fatty oil. Inaddition, dosage unit forms can contain various other materials whichmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents. The compounds can also be administeredas a component of an elixir, suspension, syrup, wafer, chewing gum orthe like. A syrup may contain, in addition to the active compounds,sucrose as a sweetening agent and certain preservatives, dyes andcolorings and flavors.

The active materials can also be mixed with other active materials whichdo not impair the desired action, or with materials that supplement thedesired action.

Solutions or suspensions used for parenteral, intradermal, subcutaneous,or topical application can include any of the following components: asterile diluent, such as water for injection, saline solution, fixedoil, a naturally occurring vegetable oil like sesame oil, coconut oil,peanut oil, cottonseed oil, etc. or a synthetic fatty vehicle like ethyloleate or the like, polyethylene glycol, glycerine, propylene glycol orother synthetic solvent; antimicrobial agents, such as benzyl alcoholand methyl parabens; antioxidants, such as ascorbic acid and sodiumbisulfite; chelating agents, such as ethylenediaminetetraacetic acid(EDTA); buffers, such as acetates, citrates and phosphates; and agentsfor the adjustment of tonicity such as sodium chloride or dextrose.Parenteral preparations can be enclosed in ampules, disposable syringesor multiple dose vials made of glass, plastic or other suitablematerial. Buffers, preservatives, antioxidants and the like can beincorporated as required.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropyleneglycol and mixtures thereof. Liposomalsuspensions, including tissue-targeted liposomes, may also be suitableas pharmaceutically acceptable carriers. These may be prepared accordingto methods known to those skilled in the art. For example, liposomeformulations may be prepared as described in U.S. Pat. No. 4,522,811.

The active compounds may be prepared with carriers that protect thecompound against rapid elimination from the body, such as time releaseformulations or coatings. Such carriers include controlled releaseformulations, such as, but not limited to, implants andmicroencapsulated delivery systems, and biodegradable, biocompatiblepolymers, such as collagen, ethylene vinyl acetate, polyanhydrides,polyglycolic acid, polyorthoesters, polylactic acid and others. Methodsfor preparation of such formulations are known to those skilled in theart.

The compounds may be formulated for local or topical application, suchas for topical application to the skin and mucous membranes, such as inthe eye, in the form of gels, creams, and lotions and for application tothe eye or for intracisternal or intraspinal application. Suchsolutions, may be formulated as 0.01%-100% (weight to volume) isotonicsolutions, pH about 5-7, with appropriate salts. The compounds may beformulated as aeorsols for topical application, such as by inhalationsee, e.g., U.S. Pat. Nos. 4,044,126, 4,41 4,209, and 4,364,923!.

Finally, the compounds may be packaged as articles of manufacturecontaining packaging material, an acceptable composition containing acompound of formulae (I) and (II) provided herein, which is effectivefor treating the particular disorder, and a label that indicates thatthe compound or salt thereof is used for treating the disorder.

E. Methods of use

The compounds for use in the the methods herein have the formulae (I)and (II): ##STR5## or the hydrates and isosteres, diastereomeric isomersand mixtures thereof, or pharmaceutically acceptable salts thereof inwhich R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R_(A), R_(B), X, Q and n areselected from among (i), (ii), (iii), (iv), (v), (vi), (vii) or (viii),as described above.

These compounds have pharmacological utility and also utility asreagents. It is recognized in this art that compounds that exhibitactivities in assays that assess the ability of the compounds to alteror modulate the activity of proteins associated with the deposition ofcerebral amyloid, are pharmacologically useful and potentiallytherapeutically useful in the treatment of disorders that involve suchdeposition.

The dose ranges, which can be established empirically, for use in thetreatment of disease states will depend upon the etiology, nature, andseverity of the disease state as well as such other factors asdetermined by the attending physcian. The broad range for effectivetreatment is about 0.01 to 10 mg per kilogram (kg) of body weight perday. The preferred range is about 0.1 to 10 mg/kg of body weight perday.

The active compounds can be administered by any appropriate route, forexample, orally, parenterally, intravenously, intradermally,subcutaneously, or topically, in liquid, semi-liquid or solid form andare formulated in a manner suitable for each route of administration.Preferred modes of administration include oral and parentoral modes ofadministration.

Since, it is feasible to indirectly measure the presence of, and overthe course of time, to determine the rate of increase of those proteinsegments believed to be critical to the formation of amyloid plaqueslocated in the brain (see, e.g., U.S. Pat. No. 5,270,165 and the CSFassay provided herein and described above and in the EXAMPLES), dosagescan be empirically determined by the physician. As these techniquesinvolve the use of cerebrospinal fluids, such techniques, and otherequivalently functioning procedures, will be useful to the attendingphysician in determining the need to modify the dosage for individualpatients.

In treating these disease states, it is sufficient to start treating thepatient as soon as the attending physician makes his or her diagnosisthat the patient is suffering from one of these diseases. Thus, althoughthe progress of treatment of the patient may be monitored by themeasurements of those biological factors which characterize thediseases, it is not necessary to so-evaluate such characteristics beforetreatment. Rather it is within the provence of the attending physicianto determine when it is in the best interest of the patient to starttreatment. Therefore, patients showing increased probabilities of thedisease state, (e.g. by carrying known familial genetic markers thatincrease the probability of the incidence of neurodegenerative diseasesas well as the patient's general behavioral characteristics and otherindicia of these diseases) can be treated by the methods and with thecompositions provided herein.

1. Treatment of neurodegenerative diseases

Amyloid plaques are believed to accompany and/or be involved in theprocess responsible for the development and progression of certainneurodegenerative disease states. Without being bound by any theory ofaction, it is believed that the compounds provided herein modulate thegeneration of amyloidogenic peptides to effectuate a beneficial result.Without any intent to limit -or restrict- the compounds and methodsprovided herein to any specific mechanism of action for the end-useapplications, it is believed that the compounds effectuate a modulationof the processing of the amyloid precursor protein (APP), the progenitorof the deposited amyloidogenic Aβ peptides (39 to 43 amino acidresidues) found in senile plaques in the brains of patients diagnosedwith, for example, Alzheimer's disease. Thus, the compounds providedherein are useful in the treatment of such neurodegenerative diseasestates in which such amyloid plaques accumulate or are implicated in theetiology thereof, including, but not limited to: Alzheimer's disease,cognition deficits, Down's Syndrome, Parkinson's disease, cerebralhemorrhage with amyloidosis, dementia pugilistica, head trauma and inthe treatment of conditions characterized by a degradation of theneuronal cytoskeleton resulting from a thromo bolytic or hemorrhagicstroke.

For example, it is believed that the compounds can be used in thetreatment of Alzheimer's patients through the modulation of APPprocessing to effectuate a beneficial result by: (a) decreasing theformation of Aβ; (b) modulating the generation of a mutually exclusive,alternative-processed form of APP that precludes Aβ formation (α-sAPP);and/or, (c) modulating the generation of partially processed C-terminalAβ-containing amyloidogenic peptides.

In addition, these compounds may also beneficially modulateneurodegenerative abnormalities not thought to be associated withamyloid plaques, such as stroke, by beneficially affecting the rate ofdegeneration of the neuronal cytoskeleton that occurs as a result ofthrombolytic or hemorrhagic stroke.

It is believed that the treatment of patients with such disorders withthese compounds will result in a beneficial modulation of the causativefactors involved in neurodegenerative disease states and will result inan enhanced lifestyle as well as to delay or obviate the need toinstitutionalize these patients.

The compounds can be administered to patients in need of such treatmentin a dosage range of 0.01-10 mg per kg of body weight per day and can beadministered by any appropriate route, for example, orally,parenterally, intravenously, intradermally, subcutaneously, ortopically, in liquid, semi-liquid or solid form and are formulated in amanner suitable for each route of administration. As stated above, thedose will vary depending on severity of disease, weight of patient andother factors which a person skilled in the art will recognize.

Patients include those with a neurodegenerative disease, including butnot limited to Alzheimer's disease, cognition deficits, Down's Syndrome,Parkinson's disease, cerebral hemorrhage with amyloidosis, dementiapugilistica, and head trauma. Treatment is effected by administering tosuch patient a therapeutically effective amount of a compound of theformulae (I) and (II) defined as above. Particularly preferred for usein these methods are the compounds particularly provided herein offormulae (I) and (II) but with the proviso that, when the compounds haveformula (I): (1) at least one of the amino acid residues in theresulting tripeptide is a non-naturally-occurring α-amino acid or atleast one of the R₁, R₃ and R₅ is not a side chain of anaturally-ocurring amino acid; and (2) when X is an aldehyde, thenon-naturally occurring amino acid (or side chain thereof) is other thannorleucine or norvaline, and when the compounds have formula (II) and Xis an aldehyde, R₁ is not be norleucine or norvaline.

In some embodiments the compounds of formula (II) are also selected suchthat: (1) at least one the amino acid residues in the resultingdi-peptide is a non-naturally-occurring α-amino acid or at least one ofthe R₁ and R₃ is not a side chain of a naturally-ocurring amino acid;and (2) when X is an aldehyde, the non-naturally occurring amino acid(or side chain thereof) is other than norleucine or non/aline.

In certain preferred embodiments, the compounds have formulae I or II,particularly formula I, as defined above, but with the proviso that: (1)at least one of the amino acid residues in the resulting di ortri-peptide is a non-naturally-occurring α-amino acid or at least one ofthe R₁, R₃ and R₅ is not a side chain of a naturally-ocurring aminoacid; and (2) when R₁ is the side chain from a non-naturally occuringamino acid, it is not the side chain of norleucine or norvaline.

In other preferred embodiments, the compounds have formulae I or II,particularly formula I, as defined above, but with the proviso that: (1)at least one of the amino acid residues in the resulting di ortri-peptide is a non-naturally-occurring α-amino acid or at least one ofthe R₁, R₃ and R₅ is not a side chain of a naturally-ocurring aminoacid; and (2) none of R₁, R₃ and R₅ is the side chain of norleucine ornorvaline.

2. Treatment of diseases characterized by degeneration of thecytoskeleton

Also provided are methods of treating a patient suffering from a diseasestate characterized by the degeneration oF the cytoskeleton arising froma thrombolytic or hemorrhagic stroke by administering a therapeuticallyeffective amount of a compound of the formulae (I) and (II),particularly the compounds of formulae (I), with the proviso that whenthe compounds have formula (I): (1) at least one of the amino acidresidues in the resulting tri-peptide is a non-naturally-occurringα-amino acid or at least one of the R₁, R₃ and R₅ is not a side chain ofa naturally-ocurring amino acid; and (2) when X is an aldehyde, thenon-naturally occurring amino acid (or side chain thereof) is other thannorleucine or norvaline, and when the compounds have formula (11) and Xis an aldehyde, R₁ cannot be norleucine or norvaline.

In certain preferred embodiments, the compounds for use in this methodof treatment have formulae I or II, particularly formula I, as definedabove, but with the proviso that (1) at least one of the amino acidresidues in the resulting tri-peptide is a non-naturally-occurringα-amino acid or at least one of the R₁, R₃ and R₅ is not a side chain ofa naturally-ocurring amino acid; and (2) when R₁ is the side chain froma non-naturally occuring amino acid, it is not the side chain ofnorleucine or norvaline.

In some embodiments the compounds of formula (II) are also selected suchthat: (1) at least one of the amino acid residues in the resultingdipeptide is a non-naturally-occurring α-amino acid or at least one ofthe R₁ and R₃ is not a side chain of a naturally-ocurring amino acid;and (2) when X is an aldehyde, the non-naturally occurring amino acid(or side chain thereof) is other than norleucine or norvaline.

In other preferred embodiments, the compounds have formulae I or II,particularly formula I, as defined above, but with the proviso that: (1)at least one of the amino acid residues in the resulting di ortri-peptide is a non-naturally-occurring α-amino acid or at least one ofthe R₁, R₃ and R₅ is not a side chain of a naturally-ocurring aminoacid; and (2) none of R₁, R₃ and R₅ is the side chain of norleucine ornorvaline.

3. Protease inhibition

The compounds provided herein have activity as inhibitors of proteases,such cysteine proteases, including calpain. It is believed by those ofskill in this art that excessive activation of the Ca²⁺ -dependentprotease calpain plays a role in the pathology of a variety ofdisorders, including cerebral ischaemia, cataract, myocardial ischaemia,muscular dystrophy and platelet aggregation. Thus, compounds that haveactivity as calpain inhibitors are considered by those of skill in thisart to be useful see, e.g., U.S. Pat. No. 5,081,284, Sherwood et al.(1993) Proc. Natl. Aced. Sci. U.S.A. 90:3353-3357!. Assays that measurethe anti-calpain activity of selected compounds are known to those ofskill in the art (see, e.g., U.S. Pat. No. 5,081,284). Activities ofinhibitors in such in vitro assays at concentrations (IC₅₀) in thenanomolar range or lower are indicative of therapeutic activity. Suchcompounds also have utility in the purification of proteinases, such ascysteine proteases, on affinity columns of these compounds (see, U.S.Pat. No. 5,081,284). Also, calpain inhibitors, such asN-Acetylleucylleucylnorleucinal see, e.g., EP 0 504 938 A2; and Sherwoodet al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:3353-3357!, which iscommercially available, are used as reagents in the study of proteintrafficking and other cellular processes see, e.g., Sharma et al. (1992)J. Biol. Chem. 267:5731-5734!. Finally, inhibitors of cysteine proteasesstrongly inhibit the growth of Plasmodium falciparum and Schistosomamansoni see, e.g., Scheibel et al. (1984) Protease inhibitors andantimalarial effects. In: Malaria and the Red Cell, Progress in CLinicaland Biolgoical Research, Alan R. Liss, Inc., New York, pp. 131-142!.

The following specific examples further illustrate the methods by whichcompounds of formulae I and II may be prepared but are not meant tolimit the scope of this invention to the specific compounds. Thus, thefollowing examples are included for illustrative purposes only and arenot intended to limit the scope of the invention.

EXAMPLE 1 Preparation of N-L-(methyl)Nle N-methoxy-N-methylamidehydrochloride

To a stirred solution of N-Boc-L-Nle-OH (5.0 g, 22.0 mmol) in anhydrousmethylene chloride (CH₂ Cl₂) (25 mL) under Argon (Ar) at roomtemperature (R.T.) were added successively hydroxybenzotriazole hydrate(HOBT) (5.8 g, 43.0 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (EDC) (4.19, 22.0 mmol) N,O-dimethylhydroxylaminehydrochloride (1.9 g, 20.0 mmol) and triethylamine (Et₃ N) (3 mL, 22.0mmol). The reaction mixture was stirred for 16 h. Additional CH₂ Cl₂ (25mL) was added and the mixture was washed with saturated aqueous sodiumhydrogen carbonate (sat. NaHCO₃) (2×10 mL), 10% aqueous hydrogenchloride (10% HCl) (2×10 mL), saturated aqueous sodium chloride (sat.NaCl) (2×10 mL), dried over anhydrous magnesium sulfate (MgSO₄),filtered and concentrated. The resultant residue was purified by flashchromatography on silica gel (ethyl acetate: hexane (EA:H); 1:3) to giveN-Boc-L-Nle N-methoxy-N-methylamide as a colorless oil (4.1 g, 69.3%): ¹H NMR (CDCl₃, 300 MHz) δ 0.87-0.92 (m, 3H), 1.24-1.72 (m, 15H), 3.21 (s,3H), 3.78 (s, 3H), 4.66-4.68 (m, 1H), 5.16 (d, 1H, J=9.0 Hz) ppm.

To a stirred solution of the N-Boc-L-Nle N-methoxy-N-methylamide (1.59g, 5.5 mmol) in 20:1 anhydrous tetrahydrofuran: dimethylformamide(THF:DMF) (22 mL) at 0° C. under Ar was added methyl iodide (0.68 mL,11.0 mmol) and 60% sodium hydride (NaH) in oil dispersion (0.25 g, 6.0mmol). The reaction mixture was refluxed for 16 h. The mixture waspoured onto 10% HCl and was extracted with EA (3×20 mL). The combinedorganic extracts were washed with sat. NaCl (2×10 mL), dried (MgSO₄),filtered and concentrated in vacuo to afford N-Boc-L-(methyl)NleN-methoxy-N-methylamide derivative as a yellow oil (0.86 g, 54.5%): ¹ HNMR (CDCl₃, 300 MHz) δ 0.87-0.93 (m, 3H), 1.25-1.48 (m, 13H), 1.71-1.73(m, 2H), 2.83-2.85 (m, 3H), 3.19 (s, 3H), 3.69-3.74 (d, 3H, J=15.0 Hz),4.80-4.95 (broad (b)-m, 1H) ppm.

N-Boc- L-(methyl)Nle N-methoxy-N-methylamide (0.84 g, 2.9 mmol) wastreated with 4N HCl in dioxane (15 mL) at R.T. The reaction mixture wasstirred for 1.5 h then concentrated in vacuo. The solid was treated withanhydrous ether (3×10 mL) and concentrated in vacuo. The resultingN-L-(methyl)Nle N-methoxy-N-methylamide hydrochloride was isolated as awhite solid (0.47 g, 85.7%): ¹ H NMR (CDCl₃, 300 MHz) δ 0.91-0.95 (m,3H), 1.73 (m, 4H), 2.06-2.08 (m, 2H), 2.27-2.29 (m, 6H), 3.71-4.09 (m,3H) ppm.

EXAMPLE 2 Preparation of N-Cbz-L-Leu-L-Leu-DL-(methyl)norleucinal

To a stirred solution of N-L-(methyl)Nle N-methoxy-N-methylamidehydrochloride isolated above in Example 1 (0.87 g, 2.3 mmol) inanhydrous CH₂ Cl₂ (20 mL) under Ar at R.T. were added HOBT (0.62 g, 4.6mmol), EDC (0.44 g, 2.3 mmol), N-Cbz-L-Leu-L-Leu-OH (0.47 g, 2.1 mmol)and Et₃ N (0.3 mL, 2.3 mmol). The reaction mixture was stirred for 16 hthen taken up in additional CH₂ Cl₂ (20 mL). The mixture was washed withsat. NaHCO₃ (2×10 mL), 10% HCl (2×10 mL), sat. NaCl (2×10 mL), dried(MgSO₄), filtered and concentrated. The resulting crude was purified byflash chromatography on silica gel (EA:H, 1:3) to yieldN-Cbz-L-Leu-L-Leu- L-(methyl)Nle N-methoxy-N-methylamide as a colorlessoil (0.4 g, 34.9%): ¹ H NMR (CDCl₃, 300 MHz) δ 0.87-1.01 (m, 3H),1.16-1.95 (m, 12H), 2.95-3.21 (m, 6H), 3.65-3.82 (m, 3H), 4.23 (m,1H),4.97-5.52 (m, 5H), 6.72-6.75 (m, 1H), 7.30-7.36 (m, 5H) ppm.

To a stirred solution of N-Cbz-L-Leu-L-Leu-L-(methyl)NleN-methyloxy-N-methylamide (0.33 g, 0.55 mmol) in anhydrous THF (3 mL)under Ar at -78° C. was added a solution of 1.0M diiso-butylaluminumhydride (DIBAL) in hexanes (2.7 mL, 2.7 mmol). The reaction mixture wasstirred for 1 h at -78° C. then quenched by adding a saturated aqueousRochelle salt solution (15 mL). The mixture was extracted with EA (3×20mL). The combined organic extracts were dried (MgSO₄), filtered,concentrated and purified on silica gel (EA:H; 2:3) to afford the titlecompound N-Cbz-L-Leu-L-Leu-DL-(methyl)norleucinal as a colorless oil(0.12 g, 45%): Reporting a mixture of diastereomers ¹ H NMR (CDCl₃, 300MHz) δ 0.93-1.01 (overlapping (ol)-m, 15H), 1.25-1.71 (ol-m, 12H), 4.24(ol-m, 1H),4.52 (ol-m, 1H), 5.03-5.13 (ol-m, 3H), 7.32-7.36 (ol-m, 5H),9.50-9.56 (ol-m, 1H) ppm.

EXAMPLE 3 Preparation of N-Ac-L-Leu-L-Leu-DL-cyclohexylalaninal

To a stirred solution of N-Ac-L-Leu-OH (5.0 g, 28.9 mmol) in anhydrousCH₂ Cl₂ (50 mL) at R.T. under Ar were added HOBT (7.9 g, 57.7 mmol), EDC(5.5 g, 28.9 mmol), H-L-Leu-OBn p-TsOH (9.9 g, 24.9 mmol) and Et₃ N (4mL, 28.9 mmol). The reaction mixture was stirred for 16 h then taken upin additional CH₂ Cl₂ (20 mL). The organic was washed with sat. NaHCO₃(2×10 mL), 1N HCl (2×10 mL), sat. NaCl (2×10 mL), dried (MgSO₄),filtered and concentrated to afford N-Ac-L-Leu-L-Leu-OBn as an oil (9.0g, 96%): ¹ H NMR (CDCl₃, 300 MHz) δ 0.87-0.90 (m, 12H), 1.43-1.68 (m,6H), 1.94,+1.96 (s+s, 3H), 4.51-4.63 (m, 2H), 5.09-5.19 (m, 2H),6.61-6.64 (d, 1H, J=9.0 Hz), 6.95-6.97 (d, 1H, J=6.0 Hz), 7.28-7.39 (m,5H) ppm.

To a stirred solution of N-Ac-L-Leu-L-Leu-OBn (9.09 g, 23.9 mmol) in EA(100 mL) at R.T. was added 20% palladium hydroxide on carbon (Pd (OH)₂/C) (0.9 g, 10% by weight). The reaction mixture was placed under 20 psihydrogen (H₂). After 1 h the reaction mixture was filtered throughcelite and concentrated in vacuo to give N-Ac-L-Leu-L-Leu-OH as a whitesolid (6.0 g, 87.67%): ¹ H NMR (300 MHz, CDCl₃) δ 0.82-0.90 (m, 12H),1.39-1.82 (m, 9H), 4.18-4.34 (m, 2H), 7.94-8.13 (m, 2H) ppm.

To a stirred solution of N-Ac-L-Leu-L-Leu-OH (1.0 g, 3.5 mmol) inanhydrous CH₂ Cl₂ (10 mL) at R.T. under Ar was added1,1-carbonyldiimidazole (CDl) (0.68 g, 4.2 mmol). Then Et₃ N (1.0 mL,7.0 mmol) and cyclohexylalanine hydrochloride (0.73 g, 3.5 mmol) weresuccessively added. After 16 h of stirring the reaction mixture wasconcentrated. The residue was tritrated with 1N aqueous hydrogenchloride (1N HCl), washed with water and dried in vacuo to afford theN-Ac-L-Leu-L-Leu-L-Ala(cyclohexyl)-OH acid as a white solid (0.66 g,42.5%): ¹ H NMR (CDCl₃, 300 MHz) δ 0.90-0.98 (m, 12H), 1.15-1.82 (m,19H), 1.95-1.98 (m, 3H), 4,36-4.46 (m, 3H) ppm. To a stirred solution ofN-Ac-L-Leu-L-Leu-L-Ala(cyclohexyl)-OH (0.6 g, 1.2 mmol) in anhydrous DMF(10 mL) at R.T. under Ar were added HOBT (0.26 g, 1.3 mmol),4-dimethylaminopyridine (DMAP) (0.05 g) and 1-thiol propane (0.12 mL,1.3 mmol). The reaction mixture was stirred for 16 h, then poured ontoEA/10% HCl (30 mL/10 mL). The organic layer was separated and washedwith sat. NaCl (2×10 mL), dried (MgSO₄), filtered and concentrated.Purification by flash chromatography on silica gel (EA:H; 1:3) affordedthe N-Ac-L-Leu-Leu-L-Ala(cyclohexyl)-SCH₂ CH₂ CH₃ as a white solid (0.3g, 44.4%): ¹ H NMR (CDCl₃, 300 MHz) δ 0.86-0.98 (m, 15H), 1.49-2.07 (m,24H), 2.76-2.87 (m, 2H), 4.64-4.80 (m, 3H) ppm.

To a solution of the thiol ester (0.27 g, 0.52 mmol) in anhydrous CH₂Cl₂ at R.T. under Ar were added 10% palladium on carbon (Pd/C) (27 mg,10% by weight) and triethylsilane (Et₃ SiH) (0.43 mL, 2.6 mmol). Themixture was stirred for 16 h, then filtered through celite andconcentrated. The residue was purified by flash chromatography on silicagel (EA:H; 1:3→EA) to afford the title compoundN-Ac-L-Leu-L-Leu-DL-cyclohexylalaninal as a white solid (0.12 g,54.79%): Reporting a mixture of diastereomers ¹ H NMR (CDCl₃, 300 MHz) δ0.85-1.98 (ol-m, 31H), 1.97+1.98 (s+s, 3H), 4.25-4.38 (ol-m, 2H),4.60-4.78 (ol-m, 1H), 7.00-8.00 (ol-m, 3H), 9.48+9.54 (s+s, 1H) ppm.

EXAMPLE 4 Preparation of (2S)-N-Ac-L-Leu-L-Leu N-2-(hexanenitrile)!amide

To a stirred solution of N-Boc-L-Nle-OH (0.5 g, 2.2 mmol) in anhydrousTHF (15 mL) under Ar at -23° C. was added 4-methylmorpholine followed byiso-butyl chloroformate (0.21 mL, 2.2 mmol). The reaction mixture wasthen treated with gaseous NH₃ for 1 h. Stirring was continued at -23° C.for an additional 2 h. The mixture was warmed to R.T. and then pouredonto 10% aqueous citric acid (20 mL). The mixture was extracted with EA(3×50 mL). The combined organics were washed with sat. NaHCO₃ (3×25 mL),sat. NaCl (2×25 mL), dried (MgSO₄), filtered and concentrated to affordN-Boc-L-Nle-NH₂ as a white solid (0.48 g, 91%): ¹ H NMR (CDCl₃, 300 MHz)δ 0.84-0.92 (m, 3H), 1.22-1.31 (m, 4H), 1.41 (s, 9H), 1.49-1.59 (m, 1H),1.74-2.01 (m, 1H), 4.03-4.1 (m, 1H), 5.06 (d, 1H), 5.75 (s, 1H), 6.25(s, 1H) ppm.

To a stirred solution of the amide (N-Boc-L-Nle-NH₂) (0.16 g, 0.67 mmol)in anhydrous CH₂ Cl₂ (4, mL) at R.T. under Ar were added anhydrouspyridine (0.27 mL, 3.4 mmol) and p-toluenesulfonyl chloride (0.26 g,1.34 mmol). The reaction mixture was stirred for 3 days, then treatedwith sat. NaHCO₃ (4 mL). The mixture was stirred for 1 h. The organicwas separated and the aqueous was extracted with CH₂ Cl₂ (2×10 mL). Thecombined organics were washed with 1N HCl (2×5 mL), dried (MgSO₄),filtered and concentrated to give a crude residue. Purification by flashchromatography on silica gel (EA) afforded(2S)-N-Boc-2-amino-hexanenitrile as an oil (0.14 g, 100%): ¹ H NMR(CDCl₃, 300 MHz) 0.93 (t, 3H, J=7.3 Hz), 1.25-1.52 (m, 13H), 1.75-1.91(m, 2H), 4.50-4.61 (m, 1H), 4.82 (bs, 1H) ppm.

To the nitrile (1.0 g, 4.71 mmol) was added 4N HCl/dioxane (10 mL). Thereaction mixture was stirred at R.T. for 30 min. The resultantprecipitate was filtered and washed with hexanes (3×5 mL) to afford(2S)-2-amino-hexanenitrile hydrochloride as a white solid (0.68 g,97.59%): ¹ H NMR (CD₃ OD, 300 MHz) δ 0.95-0.99 (m, 3H), 1.10-1.60 (m,4H), 1.89-2.00 (m, 2H), 4.20-4.30 (m, 1H) ppm.

To a stirred solution of N-Ac-L-Leu-L-Leu-OH (0.87 g, 3.0 mmol) inanhydrous CH₂ Cl₂ (50 mL) at R.T. under Ar was added EDC (0.58 g. 3.0mmol), HOBT (0.81 g, 6.0 mmol) and Et₃ N (0.41 mL). After 16 h, thereaction mixture was washed with sat. NaHCO₃ (2×10 mL), 1N HCl (2×10mL), sat. NaCl (2×10 mL), dried (MgSO₄), filtered and concentrated.Purification by flash chromatography on silica gel (EA) gave the titlecompound as a white solid (0.42 g, 35%): ¹ H NMR (CDCl₃, 300 MHz) δ0.82-0.92 (m, 15H), 1.21-1.99 (m, 12H), 2.02 (s, 3H), 4.51 (m, 1H), 4.64(m, 1H), 6.78 (m, 1H), 7.50 (m, 1H), 8.06 (m, 1H) ppm.

EXAMPLE 5 Preparation of N-Cbz-L-Leu-L-Leu-DL-Nle-CO₂ Et

To a stirred solution of N-Cbz-L-Leu-L-Leu-L-Nle-OH (1.0 eq) inanhydrous THF at R.T. under Ar is added DMAP (cat.), pyridine (1.0 eq),ethyl oxalyl chloride (2.0 eq). The mixture is gently refluxed for 3 h.The mixture is treated with water, stirred for 30 min. at R.T. and thenextracted with EA. The organic extracts are dried (MgSO₄), filtered andconcentrated to afford enol ester product as a crude residue. To astirred suspension of the crude residue (1.0 eq) in anhydrous ethanol atR.T. under Ar is added dropwise a solution of sodium ethoxide inanhydrous ethanol. The reaction mixture is stirred for 3 h. The ethanolis then removed and the residue is treated with ether. The ethersolution is washed with water, dried (MgSO₄) and evaporated to give acrude residue. This residue is purified by flash chromatography onsilica gel to give the peptide ketoester N-Cbz-L-Leu-L-Leu-DL-Nle-CO₂Et.

EXAMPLE 6 Preparation of N-Cbz-L-Leu-L-Leu-DL-Nle-CO₂ H

To a stirred solution of N-Cbz-L-Leu-L-Leu-DL-Nle-CO₂ Et (1.0 eq), asprepared in Example 5, in methanol at R.T. is added 1N NaOH (1.1 eq).The mixture is stirred for 6 h. The reaction mixture is cooled to 0° C.,acidified with 1N HCl (pH=3) and extracted with EA. The organic extractsare washed with water, dried (MgSO₄), filtered and concentrated to givea crude residue. Trituration with hexane and drying in vacuo gives thetitle compound, N-Cbz-L-Leu-L-Leu-DL-Nle-CO₂ H.

EXAMPLE 7 Preparation of N-Cbz-L-Leu-L-Leu-DL-Nle-CONHEt

To a stirred solution of N-Cbz-L-Leu-L-Leu-DL-Nle-CO₂ Et (1.0 eq), asprepared in Example 5, in anhydrous CH₂ Cl₂ at R.T. is added1,2-ethanedithiol (2.2 eq), followed by boron trifluoride etherate (0.1eq). The mixture is stirred for 16 h. Water and ethyl ether are added.The organic layer is separated, washed with water, sat. NaCl, dried(MgSO₄), filtered and concentrated to afford the protected α-ketoester.

The protected α-ketoester is dissolved in ethanol (5 mL) and cooled to0° C., and ethylamine is bubbled through the solution. The mixture iswarmed to R.T., stirred overnight, filtered and concentrated. The cruderesidue is purified by flash chromatography on silica gel to give thetitle compound N-Cbz-L-Leu-L-Leu-DL-Nle-CONHEt.

EXAMPLE 8 Preparation of the precusor ethyl2-amino-4-methyl-4-pentenoate hydrochloride

To a solution of N-(diphenylmethylene)glycine ethyl ester (6.6 g, 24.7mmol) in anhydrous THF at -78° C. under Ar was slowly added 1.0M lithiumbis(trimethylsilyl)amide (LiHMDSi) in THF (24.7 mL, 24.7 mmol) over 15min. Stirring was continued for 30 min at -78° C., then3-bromo-2-methylpropene (2.5 mL, 25.0 mmol) was added. The mixture wasgradually warmed to R.T. then stirred for 1 h at R.T. The reactionmixture was treated with water and then concentrated. The residue wastaken up in EA (50 mL). The organic layer was washed with sat. NaCl(2×10 mL), dried (MgSO₄), filtered and concentrated. The crude waspurified by flash chromatography on silica gel (EA:H; 1:4) to give ethyl4-methyl-2- (diphenylmethylene)amine!-4-pentenoate as a colorless oil(6.4 g, 89.3%): ¹ H NMR (CDCl₃, 300 MHz) δ 1.23-1.29 (t, 3H, J=6.0 HZ),1.48-1.49 (m, 3H), 2.55-2.69 (m, 2H). 4.11-4.24 (m, 3H), 4.71-4.75 (m,2H), 7.16-7.83 (m, 10H) ppm.

To a stirred solution of the above ethyl ester (6.4 g, 20.0 mmol) inanhydrous ether (15 mL) at R.T. was added 1N HCl (70 mL). After 40 min,the two phases were separated, and the aqueous layer was washed withether (3×10 mL). The aqueous layer was adjusted with 1N NaOH (pH=10),then extracted with ether (3×20 mL). The combined organic extracts weredried (MgSO₄), filtered and then adjusted with 4N HCl/dioxane (pH=3) andconcentrated in vacuo to afford ethyl 2-amino-4-methyl-4-pentenoatehydrochloride as an oil (3.11 g, 79.4%): ¹ H NMR (CDCl₃, 300 MHz) δ1.25-1.32 (t, 3H, J=6.0 Hz), 1.81 (s, 3H), 2.74-2.81 (m, 2H), 4.22-4.29(m, 2H), 4.98 (d, 2H, J=12.0 Hz) ppm.

EXAMPLE 9 Preparation of (2SR)-N-Cbz-L-Leu-L-Leu N-2-(4-methyl-4-pentenal)!amide

To a stirred solution of N-Cbz-L-Leu-OH (5.6 g, 20.7 mmol) in anhydrousCH₂ Cl₂ (50 mL) at R.T. under Ar were added successively HOBT (5.6 g,41.5 mmol), EDC (4.0 g, 20.7 mmol), H-L-Leu-OCH₃ HCl (3.4 g, 18.8 mmol)and Et₃ N (2.9 mL, 20.7 mmol). The reaction mixture was stirred for 16h. The mixture was taken up in additional CH₂ Cl₂ (30 mL), washed withsat. NaCl (2×10 mL), dried (MgSO₄), filtered and concentrated. Theresulting residue was purified by flash chromatography on silica gel(EA:H; 1:2) to afford the dipeptide N-Cbz-L-Leu-L-Leu-OCH₃ as a whitesolid (6.0 g, 64.1%): ¹ H NMR (CDCl₃, 300 MHz) δ 0.86-0.95 (ol-t, 12H,J=6.0 Hz), 1.48-1.74 (m, 6H), 3.73 (s, 3H), 4.20-4.25 (m, 1H), 4.56-4.63(m, 1H), 5.11 (s, 2H), 5.20 (d, 1H, J=6.0 Hz), 6.36 (d, 1H, J=6.0 Hz),7.31-7.39 (m, 5H) ppm.

To a stirred solution of N-Cbz-L-Leu-L-Leu-OCH₃ (6.0 g, 14.6 mmol) inMeOH/H₂ O (3:1) (60 mL) at R. T. was added lithium hydroxide monohydrate(LiOH.H₂ O) (1.0 g, 43.9 mmol) and hydrogen peroxide H₂ O₂ (30% weightin H₂ O) (4.5 mL, 43.9 mmol). The reaction mixture was stirred for 3.5 hand then quenched with 10% HCl. The resulting mixture was extracted withEA (3×20 mL). The combined organic extracts were washed with sat. NaCl(2×10 mL), dried (MgSO₄) and concentrated in vacuo to affordN-Cbz-L-Leu-L-Leu-OH as a white solid (5.0 g, 90.5%): ¹ H NMR (CDCl₃,300 MHz) δ 0.89-0.92 (t, 12H, J=3.0 Hz), 1.48-1.71 (m, 6H), 4.11-4.13(m, 1H), 4.55-4.62 (m, 1H), 5.09 (s, 2H), 5.64 (d, 1H, J=9.0 Hz), 6.83(d, 1H, J=9.0 Hz), 7.33-7.40 (m, 5H) ppm.

To a stirred solution of N-Cbz-L-Leu-L-Leu-OH (3.2 g, 8.5 mmol) inanhydrous CH₂ Cl₂ (35 mL) at R. T. under Ar were added HOBT (2.3 g, 17.1mmol), EDC (1.6 g, 8.5 mmol), ethyl 2-amino-4-methyl-4-pentenoatehydrochloride, as prepared in Example 8, (1.5 g, 7.8 mmol) and Et₃ N(1.2 mL, 8.5 mmol). The reaction mixture was stirred for 16 h at R.T.The mixture was taken up in CH₂ Cl₂ (30 mL). The organic layer waswashed with sat. NaHCO₃ (2×10 mL), 10% HCl (2×10 mL), sat. NaCl (2×10mL), dried (MgSO₄), filtered and concentrated. The residue was purifiedby flash chromatography on silica gel (EA:H; 1:4) to afford(2SR)-N-Cbz-L-Leu-L-Leu N- 2- ethyl(4-methyl-4-pentenoate)!amide as awhite solid (1.9 g, 47.3%): Reporting a mixture of diastereomers ¹ H NMR(CDCl₃, 300 MHz) δ 0.88-0.92 (ol-m, 12H), 1.21-1.29 (ol-m, 3H),1.51-1.71 (ol-m, 9H), 2.39-2.52 (ol-m, 2H), 4.13-4.21 (ol-m, 3H),4.47-4.82 (ol-m, 4H), 5.10 (ol-m, 2H), 5.32 (d, 1H, J=9.0 Hz), 6.44-6.77(m, 2H), 7.29-7.36 (ol-m, 5H) ppm.

To a stirred solution of the above ethyl ester (1.9 g, 3.6 mmol) inanhydrous THF (10 mL) at 0° C. under Ar was added lithium borohydride(LiBH₄) (0.16 g, 7.1 mmol). Stirring was continued for 30 min at 0° C.then the mixture was warmed to R. T. After 1 h, 1N HCl (1 mL) was addedto the reaction mixture, and then extracted with EA (3×20 mL). Thecombined organic extracts were washed with sat. NaCl (2×10 mL), dried(MgSO₄), filtered and concentrated/n vacuo to afford the crude residue.The residue was purified by flash chromatography on silica gel (EA:H;1:3) to yield the alcohol as a white solid (1.4 g, 85.6%): Reporting amixture of diastereomers ¹ H NMR (CDCl₃, 300 MHz) δ 0.88-0.95 (ol-m,12H), 1.47-1.73 (ol-m, 9H), 2.16-2.27 (ol-m, 2H), 3.54-3.67 (ol-m, 2H),4.12 (ol-m, 2H), 4.33-4.40 (ol-m, 1H), 4.74-4.80 (ol-m, 2H), 5.03-5.37(ol-m, 3H), 6.34-6.37 (ol-m, 2H), 7.30-7.40 (ol-m, 5H) ppm.

To a stirred solution of anhydrous dimethyl sulfoxide (DMSO) (0.18 mL,2.25 mmol) in anhydrous THF (5 mL) at -78° C. under Ar was added oxalylchloride (0.24 mL, 2.25 mmol) dropwise over 5 min. After 30 min, thereaction mixture was treated with a solution of the alcohol (0.5 g, 1.0mmol) in anhydrous THF (5 mL). Stirring was continued at -78° C. for anadditional 30 min, then Et₃ N (0.65 mL, 4.7 mmol) was added. Thereaction mixture was warmed to 0° C. and stirred for 15 min. EA (50 mL)was added to the mixture and the organic layer was washed with sat.NaHCO₃ (2×10 mL), 10% HCl (2×10 mL) and sat. NaCl (2×10 mL). The organiclayer was dried (MgSO₄), filtered and concentrated to afford a cruderesidue. Purification by flash chromatography on silica gel (kieselgel60 silanisiert) (EA:H; 1:5) yielded the title compound as a white solid(0.23 g, 47.8%): Reporting a mixture of diastereomers ¹ H NMR (CDCl₃,300 MHz) δ 0.88-0.98 (ol-m, 12H), 1.52-1.73 (ol-m, 9H), 2.29-2.58 (ol-m,2H), 4.19 (ol-m, 1H), 4.49-4.51 (ol-m, 2H), 4.76-4.86 (ol-m, 2H),5.00-5.09 (ol-m, 2H), 5.39-5.42 (ol-m, 1H), 6.70-7.14, (ol-m, 2H),7.34-7.42 (ol-m, 5H) ppm.

EXAMPLE 10 Preparation of (2S)-N-Cbz-L-Leu-L-Leu N-2-(thiazole-oxo-pentyl)!amide

To a stirred solution of thiazole (0.11 mL, 1.55 mmol) in anhydrousether (8 mL) at -78° C under Ar was slowly added 1.8M N-butyllithium inhexanes (nBuLi) (1.0 mL, 1.7 mmol). After an additional 20 min ofstirring at -78° C. N-Boc-L-Nle N-methoxy-N-methylamide (0.17 g, 0.62mmol) in anhydrous ether (5 mL) was added. Stirring was continued for 1h at -78° C. then gradually warmed to R. T. The resulting mixture wastreated with 1N HCl (1 mL), 1N NaOH (pH=9), and extracted with ether(3×10 mL). The combined organic layers were washed with sat. NaHCO₃(2×10 mL), sat. NaCl (2×10 mL), dried (MgSO₄), filtered andconcentrated. Purification by flash chromatography on silica gel (EA:H;1:5) afforded the (2S)-N-Boc-2-amino-thiazole-oxo-pentyl derivative as awhite solid (0.14 g, 76%): ¹ H NMR (CDCl₃, 300 MHz) δ 0.86 (t, 3H, J=6.0Hz), 1.23-1.62 (m, 13H), 1.66-1.75 (m, 2H), 5.32-5.46 (m, 2H), 7.70 (d,1H, J=3.0 Hz), 8.03 (d, 1H, J=6.0 Hz) ppm.

The above derivative (0.13 g, 0.44 mmol) was treated with 4N HCl/dioxane(5 mL) at R. T. After 30 min the reaction mixture was concentrated invacuo. The resulting solid was recrystallized (MeOH/Ether) to give thehydrochloride as a white solid (0.1 gr, 75%): ¹ H NMR (CD₃ OD, 300 MHz)δ 0.64-0.73 (t, 3H, J=6.0 Hz), 1.17-1.21 (m, 4H), 1.76-1.99 (m, 2H),4.86-4.91 (m, 1H), 7.72-7.98 (m, 2H) ppm.

To the resulting hydrochloride (0.09 g, 0.33 mmol) in anhydrous CH₂ Cl₂(10 mL) at R. T. under Ar was added HOBT (0.19, 0.73 mmol), EDC (0.07 g,0.36 mmol), N-Cbz-L-Leu-L-Leu-OH (0.09 g, 0.33 mmol) and Et₃ N (0.09 mL,0.66 mmol). After 16 h CH₂ Cl₂ (20 mL) was added, and the organic layerwas washed with sat. NaHCO₃ (2×10 mL), 1N HCl (2×10 mL), sat. NaCl (2×10mL), dried (MgSO₄), filtered and concentrated. Purification by flashchromatography on silica gel (EA:H; 1:1) afforded the title compound asa white solid (0.16 gr, 88%): ¹ H NMR (CDCl₃, 300 MHz) δ 0.83-1.06 (m.15H), 1.20-2.15 (m, 12H), 4.10-4.30 (m, 1H), 4.48-4.61 (m, 1H), 5.11 (s,2H), 5.15-5.25 (m, 1H), 5.60-5.70 (m, 1H), 6.32-6.50 (m, 1H), 7.30-7.40(m, 5H), 7.70 (dd, 1H, J=6.0, 3.0 Hz), 8.05 (dd, 1H, J=6.0, 3.0 Hz) ppm.

EXAMPLE 11 Preparation of N-Ac-L-Leu-L-Leu-L-Nle-COCHN₂

To a stirred solution of N-Ac-L-Leu-L-Leu-L-Nle-OH (0.3 g, 0.77 mmol),in anhydrous THF (10 mL) at -23° C. were added 4-methylmorpholine (0.1mL, 0.85 mmol) and iso-butyl chloroformate (0.11 mL, 0.85 mmol). Themixture was stirred at -23° C. for 20 min. The resulting mixture wasadded to a solution of diazomethane (10.0 mmol) in anhydrous ether (5mL) at 0° C. The mixture was stirred at 0° C. for an additional 1 h thengradually warmed to R. T., and stirred for an addition 3 h at R. T. Thereaction mixture was taken up in CH₂ Cl₂ (30 mL) and the organic layerwas washed with sat. NaHCO₃ (2×10 mL), sat. NaCl (2×10 mL), dried(MgSO₄) and concentrated. Recrystallization (EA/H) afforded the desireddiazomethyl ketone (N-Ac-L-Leu-L-Leu-L-Nle-COCHN₂) as a white solid(0.33 g, 92%): ¹ H NMR (CDCl₃, 300 MHz) δ 0.88-0.99 (m, 15H), 1.23-1.90(m, 12H), 2.00 (s, 3H), 4.53-4.68 (m, 1H), 4.72-4.79 (m, 2H), 5.86 (s,1H), 7.71 (broad doublet (bd) 1H), 7.96 (bd, 1H), 8.16 (bd, 1H) ppm.

EXAMPLE 12 Preparation of (3SR)-N-Ac-L-Leu-L-Leu N-3-(1,1,1-trifluoro-2-oxo-heptyl)!amide

To a stirred solution of 1-nitropentane (1.0 g, 8.5 mmol) andtrifluoroacetaldehyde ethyl hemiacetal (1.2 mL, 8.5 mL) was addedpotassium carbonate (K₂ CO₃) (0.06 g, 0.43 mmol). The reaction mixturewas heated at 60° C. under Ar for 3 h. The mixture was cooled to R.T.,then taken up in EA (50 mL). The organic layer was washed with 1N HCl(2×10 mL), sat. NaCl. (2×10 mL), dried (MgSO₄), filtered andconcentrated to give (2SR)-(3SR)-3-nitro-1,1,1-trifluoro-2-heptanol as acrude oil (1.7 g, 3%): ¹ H NMR (CDCl₃, 300 MHz) δ 0.91 (t, 3H, J=7.2Hz), 1.24-1.43 (m, 4H), 2.04-2.11 (m, 2H), 4.08-4.77 (m, 3H) ppm.

To a stirred solution of the nitro-alcohol derivative (1.6 g, 7.4 mmol)in methanol (10 mL) was added Raney Nickel (0.16 g, 10% by weight). Themixture was placed under 35 psi of H₂ for 16 h, and then was filteredthrough celite. The celite was washed with methanol (3×10 mL). Thecombined organics were concentrated to give(2SR)-(3SR)-3-amino-1,1,1-trifluoro-2-heptanol as an oil (0.8 g, 64.9%):¹ H NMR (CD₃ OD, 300 MHz) δ 0.89-0.95 (m, 3H), 1.20-2.10 (m, 6H),3.30-4.40 (m, 4H) ppm.

To a stirred solution of (2SR)-(3SR)-3-amino-1,1,1-trifluoro-2-heptanol(0.36 g, 2.14 mmol) in anhydrous CH₂ Cl₂ (20 mL) were addedN-Ac-Leu-Leu-OH (0.67 g, 2.4 mmol), HOBT (0.33 g, 2.4 mmol), EDC (0.46g, 2.4 mmol) and Et₂ N (0.33 mL, 2.4 mmol). The reaction mixture wasstirred for 18 h then washed with sat. NaHCO₃ (2×10 mL), 1N HCl (2×10mL), sat. NaCl (2×10 mL), dried (MgSO₄), filtered and concentrated.Purification by flash chromatography on silica gel (EA:H; 1:2) affordedthe trifluoromethyl alcohol peptide derivative as a white solid (0.85 g,88.8%): Reporting a mixture of diastereomers ¹ H NMR (DMSO-d₆, 300 MHz)δ 0.89-0.95 (ol-m, 15H), 1.05-1.90 (ol-m, 12H), 1.95 (s, 3H), 3.91-4.50(ol-m, 4H) ppm.

To a stirred solution of the above product (0.20 g, 0.44 mmol) at R. T.under Ar (0.2 g, 0.44 mmol) in anhydrous 1:1 THF/CH₂ Cl₂ (40 mL) wereadded trifluoroacetic acid (TFA) (0.10 mL, 1.32 mmol) and theDess-Martin reagent (0.56 g, 1.32 mmol). The reaction mixture wasstirred for 24 h then concentrated in vacuo. The residue was treatedwith a mixture of EA (20 mL), sat. NaHCO₃ (5 mL) and saturated aqueoussodium thiosulfate (sat. Na₂ S₂ O₃) (5 mL). The organic layer wasseparated and washed with a sat. NaHCO₃ (2×5 mL), sat. Na₂ S₂ O₃ (2×5mL), sat. NaCl (2×5 mL), dried (MgSO₄), filtered and concentrated. Theresidue was recrystallized (EA/H) to give the title compound as a whitesolid (0.11 g, 55%): Reporting a mixture of diastereomers ¹ H NMR(DMSO-d₆, 300 MHz) δ 0.81-0.87 (ol-m, 15H), 1.09-1.56 (ol-m, 12H), 1.98(ol-s, 3H), 3.90-4.0 (ol-m, 1H), 4.20-4.32 (ol-m, 2H) ppm.

EXAMPLE 13 Preparation of (2SR)-H-L-Leu N- 2-(ethyl4-methyl-4-pentenoate)!amide hydrochloride

To a stirred solution of ethyl 2-amino-4-methyl-4-pentenoatehydrochloride (as prepared in Example 8) (0.70 g, 3.6 mmol) in CH₂ Cl₂(10 mL) at R. T. under Ar were added N-Boc-L-Leu-OH (1.0 g, 4.0 mmol),HOBT (1.19 g, 7.9 mmol), EDC (0.76 g, 4.0 mmol) and Et₃ N (0.55 mL, 4.0mmol). The reaction mixture was taken up in additional CH₂ Cl₂ (20 mL)and washed with sat. NaHCO₃ (2×10 mL), 1N HCl (2×10 mL), sat. NaCl (2×10mL), dried (MgSO₄), filtered and concentrated. The resulting residue waspurified by flash chromatography on silica gel (EA:H; 1:1) to afford thedipeptide ethyl ester as a white solid (1.05 g, 78.6%): ¹ H NMR (CDCl₃,300 MHz) δ 0.92-0.97 (m, 6H), 1.25-1.30 (t, 3H, J=6 Hz), 1.44-1.7 (m,15H), 2.37-2.57 (m, 2H), 4.15-4.22 (m, 2H), 4.64-4.94 (m, 4H) ppm.

To the above ester (1.0 g, 2.7 mmol) was added 4N HCl/dioxane (15 mL),stirred at R.T. for 4 h, then the solvent was removed. Co-evaporationwith ether (3×5 mL) yielded the title compound as a white solid (0.8 g,96.3%): ¹ H NMR (CDCl₃, 300 MHz) δ 0.89-1.00 (m, 6H), 1.21-1.31 (m, 3H),1.44-1.84 (m, 6H), 2.34 (m, 2H), 4.16-4.75 (m, 4H) ppm.

EXAMPLE 14 Preparation of (2SR)-N-(2S)-2-benzoxy-4-methylpentanoyl!-L-Leu N- 2-(4-methyl-4-pentenal)!amide

To a stirred solution of L-Leu-OH (5.0 g, 38.2 mmol) in 1N H₂ SO₄ (50mL) at 0° C. was slowly added over 11/2 h a solution of sodium nitrite(NaNO₂) (7.5 g, 0.11 mmol) in water (20 mL) while maintaining thetemperature at 0° C. The reaction mixture was gradually warmed to R.T.,stirred for 24 h, and concentrated to give a white solid. The solid wasextracted with ether (5×50 mL). The combined organic layers were dried(MgSO₄), filtered and concentrated to give(2S)-2-hydroxy-4-methylpentanoic acid as an oil (4.1 g, 81.2%): ¹ H NMR(CDCl₃, 300 MHz) δ 0.98 (d, 6H, J=12.0 Hz), 1.57-1.67 (m, 2H), 1.82-1.93(m, 1H), 4.36 (t, 1H, J=6.0 Hz) ppm.

To a stirred solution of the acid (4.0 g, 30.5 mmol) in anhydrous DMF(20 mL) at R. T. under Ar was added cesium carbonate (Cs₂ CO₃) (12.9 g,40.0 mmol) and methyl iodide (5.7 g, 40.0 mmol). The reaction mixturewas stirred for 16 h then taken up in EA (100 mL). The organic layer waswashed with sat. NaHCO₃ (3×20 mL), 1N HCl (2×20 mL), dried (MgSO₄),filtered and concentrated. Purification by flash chromatography onsilica gel (EA:H; 1:4) gave methyl (2S)-2-hydroxy-4-methylpentanoate asa colorless oil (2.5 g, 57%): ¹ H NMR (CDCl₃, 300 MHz) δ 0.94-1.01 (m,6H), 1.56-1.74 (m, 2H), 1.87-1.96 (m, 1H), 3.79 (s, 3H), 4.24 (q, 1H,J=6.0 Hz) ppm.

To a stirred solution of methyl (2S)-2-hydroxy-4-methylpentanoate (0.5g, 3.4 mmol) in anhydrous CH₂ Cl₂ (10 mL) at R.T. under Ar was addedbenzyl 2,2,2-trichloroacetimidate (1.4 mL, 6.8 mmol) andtrifluoromethylsulfonyl acid (25 μl). After 30 min the reaction mixturewas taken up in CH₂ Cl₂ (20 mL). The organic layer was washed with sat.NaCl (2×10 mL), 1N HCl (2×10 mL), sat. NaCl (2×10 mL), dried (MgSO₄),filtered and concentrated. Purification by flash chromatography onsilica gel (EA:H; 1:10) afforded methyl(2S)-2-benzoxy-4-methylpentanoate as an oil (0.6 g, 73.5%): ¹ H NMR(CDCl₃, 300 MHz) δ 0.80-0.98 (m, 6H), 1.40-1.58 (m, 1H), 1.69-1.87 (m,2H), 3.74 (s, 3H), 3.93-4.06 (m, 1H), 4.42 (d, 1H, J=12.0 Hz), 4.68-4.80(m, 1H), 7.14-7.32 (m, 5H) ppm.

To the above methyl (2S)-2-benzoxy-4-methylpentanoate (0.69 g, 2.92mmol) in MeOH/H₂ O (5 mL/1 mL) was added LiOH.H₂ O (0.28 g, 11.7 mmol)and 30% H₂ O₂ (0.3 mL, 11.7 mmol). After stirring the reaction mixturefor 24 h, the mixture was treated with 1N HCl (pH=3) and the methanolwas removed in vacuo. The aqueous layer was extracted with EA (4×15 mL).The combined organic layers were washed with 1N HCl (2×10 mL), sat. NaCl(2×10 mL), dried (MgSO₄), filtered and concentrated.(2S)-2-benzoxy-4-methylpentanoic acid was isolated as a colorless oil(0.65 g, 100%): ¹ H NMR (CDCl₃, 300 MHz) δ 0.82-1.60 (m, 6H), 1.53-1.62(m, 1H), 1.73-1.90 (m, 2H), 3.99-4.50 (m, 1H), 4.46 (d, 1H, J=12.0 Hz),4.72 (d, 1H, J=12.0 Hz) 7.10-7.26 (m, 5H) ppm.

To a solution of the product from Example 13 (0.5 g, 1.63 mmol) inanhydrous CH₂ Cl₂ (10 mL) at R.T. under Ar was added the above acid (0.4g, 1.8 mL), HOBT (0.24 9, 1.8 mmol), EDC (0.35 g, 1.8 mmol) and Et₃ N(0.25 mL, 1.8 mmol). After 16 h the reaction mixture was washed withsat. NaHCO₃ (2×10 mL), 1N HCl (2×10 ml), sat. NaCl (2×10 mL) dried(MgSO₄), filtered and concentrated. Purification by flash chromatographyafforded the ethyl ester (0.5 g, 62.5%): Reporting a mixture ofdiastereomers ¹ H NMR (CDCl₃, 300 MHz) a 0.76 (t, 3H, J=6.0 Hz),0.91-0.99 (ol-m, 12H), 1.22-1.9 (ol-m, 9H), 2.05-2.18 (ol-m, 1H),2.34-2.43 (ol-m, 1H), 3.71-4.95 (ol-m, 9H), 7.20-7.38 (ol-m, 5H) ppm.

To a solution of the ester (0.5 g, 1.0 mmol) in anhydrous THF (10 mL) atR.T. under Ar was added LiBH₄ (0.02g, 1.0 mmol). Reaction mixture wasstirred for 4 h then quenched by the addition of 1N HCl (1 mL),concentrated, and extracted with EA (3×10 mL). The combined organiclayers were washed with sat. NaHCO₃ (2×10 mL), 1N HCl (2×10 mL), sat.NaCl (2×10 mL), dried (MgSO₄), filtered and concentrated. Purificationby flash chromatography on silica gel (EA:H; 1:3) gave the alcohol as anoil (0.13 g, 30%): Reporting a mixture of diastereomers ¹ H NMR (CDCl₃,300 MHz) δ 0.72-0.98 (ol-m, 2H), 1.10-2.21 (ol-m, 11H), 3.58-4.92 (ol-m,9H), 7.0-7.50 (ol-m, 7H) ppm.

To anhydrous DMSO (51.1 μl, 0.72 mmol) in anhydrous THF (10 mL) at -78°C. under Ar was slowly added oxalyl chloride (39.3 μl, 0.45 mmol). Themixture was stirred at -78° C. under Ar for 30 min. The alcohol (0.13 g,3.0 mmol) in anhydrous THF (5 mL) was slowly added, and stirred for anadditional 11/2 h. Et₃ N (0.18 mL, 1.4 mmol) was then added, and mixturewas gradually warmed to R.T. Stirring was continued for 2 h at R.T. Thereaction mixture was taken up in EA (50 mL). The organic layer waswashed with sat. NaCl (2×10 mL), 1N HCl (2×10 mL), sat. NaHCO₃ (2×10mL), sat. NaCl (2×10 mL), dried (MgSO₄), and concentrated. Purificationby flash chromatography on silica gel (EA:H; 1:4) afforded the desiredtitle aldehyde (0.13 g, 99%): Reporting a mixture of diastereomers ¹ HNMR (CDCl₃, 300 MHz) δ 0.72-0.91 (ol-m, 12H), 1.10-2.30 (ol-m, 11H),4.0-5.01 (ol-m, 7H), 7.0-7.30 (ol-m, 7H), 9.8+9.76 (s+s, 1H) ppm.

EXAMPLE 15 Preparation of (2SR)-(3S)-N-Cbz-L-Leu-Leu N-3-(2-hydroxy-heptanoic acid)!amide

To a stirred solution of N-Boc-L-Nle N-methoxy-N-methylamide (1.0 eq) inanhydrous THF (10 mL) at 0° C. under Ar is added LiAlH₄ (1.0 eq), andstirred for 3 h at 0° C. followed by the addition of 1N HCl (1 mL). Themixture is taken up in EA then washed with sat. NaHCO₃, 1N HCl, sat.NaCl, dried (MgSO₄), filtered and concentrated to afford theN-Boc-L-norleucinal as a crude residue. To the residue is added anice-cold solution of NaHSO₃ (8 eq) and the mixture is stirred for 24 hat 5° C. To the resulting suspension is added EA and an aqueouspotassium cyanide solution (KCN) (8 eq). The reaction mixture is stirredat R.T. for 4 h. The organic phase is washed with water and concentratedto give the cyanohydrin.

The cyanohydrin is hydrolyzed in 4N HCl/dioxane under reflux for 12 h.The solvent is removed and the residue is washed with anhydrous ether togive the hydrolyzate. To a stirred solution of N-Cbz-L-Leu-Leu-OH (1.0eq) in anhydrous CH₂ Cl₂ under Ar at R.T. are added CDl (1.1 eq). After30 min of stirring Et₃ N (2 eq) and the hydrolyzate (1.0 eq) is added.The mixture is stirred for 6 h, then concentrated. The residue istrituated with 1N HCl washed with water and dried in vacuo to afford thetitle compound.

EXAMPLE 16 Preparation of (2SR)-(3S)-N-Cbz-L-Leu-L-Leu N- 3-(methyl2-hydroxy-heptanoate)!amide

To the product obtained from Example 15 in anhydrous ether at 0° C. isadded diazomethane. After 3 h the solvent is removed and the residue ispurified by flash chromatography on silica gel to give the desiredproduct.

EXAMPLE 17 Preparation of (2SR)-(3S)-N-Cbz-L-Leu-L-Leu N- 3-(benzyl2-hydroxyheptamide)!amide

To the product of Example 15 in anhydrous CH₂ Cl₂ under Ar at R.T. isadded HOBT (1.0 eq), EDC (1.0 eq), Et₃ N (1.0 eq) and benzylamine (1.0eq). After 6 h the reaction mixture is washed with sat. NaHCO₃, sat. 1NHCl, sat. NaCl, dried (MgSO₄), filtered and concentrated. The residue ispurified by flash chromatography on silica gel to afford the desiredproduct.

EXAMPLE 18 Preparation of (3SR) (4S-N-Cbz-L-Leu-L-Leu N- 4-(benzyl3-hydroxyoctamide)!amide

To a solution of N-Boc-L-norleucinal (1.0 eq), prepared by reducingN-Boc-L-Nle N-methoxy-N-methylamide as described in Example 15, in THFat -78° C. under Ar is added ethyl lithioacetate (2.2 eq) prepared insitu by the addition of nBuLi (2.2 eq) to excess anhydrous ethylacetate. After 3 h, the reaction mixture is treated with 1N HCl, and theorganic layer is washed with 1N HCl, sat. NaHCO₃. sat. NaCl, dried (MgSO₄), filtered and concentrated. Purification by flash chromatography onsilica gel gives the ester.

The ester is treated with 4N HCl/dioxane for 30 min, and thenconcentrated in vacuo. The resulting solid is taken up in anhydrousether and concentrated in vacuo to give the hydrochloride. Thehydrochloride is used without further purification in the next step.

To the hydrochloride (1.0 eq) in anhydrous CH₂ Cl₂ at R.T. under Ar isadded HOBT (2.0 eq), EDC (1.0 eq), Et₃ N (1.0 eq) and N-Cbz-L-Leu-Leu-OH(1.1 eq). After 6 h, the organic layer is washed with sat. NaHCO₃, 1NHCl, dried (MgSO₄), filtered and concentrated. Purification by flashchromatography on silica gel gives the ester.

To a stirred solution of the above ester (1.0 eq) in MeOH-H₂ O is addedLiOH.H₂ O (2 eq) and H₂ O₂ (1.0 eq). After 4 h the reaction is quenchedby the addition of 10% HCl and then extracted with EA (2×). The combinedorganic layers are washed with sat. NaCl, dried (MgSO₄) and concentratedto give the acid.

To a solution of the acid (1.0 eq) in anhydrous CH₂ Cl₂ at R.T. under Aris added EDC (1.0 eq), HOBT (1.0 eq), Et₃ N (1.0 eq) and benzylamine(1.1 eq). The reaction mixture is stirred for 3 h, washed with sat.NaHCO₃, 1N HCl, sat. NaCl, dried (MgSO₄), filtered and concentrated.Purification by flash chromatography on silica gel affords the titlecompound.

EXAMPLE 19 Preparation of (3S)-N-Cbz-L-Leu-L-Leu N-3-(1-furfylthio-2-oxo-heptane)!amide

To the N-Boc-L-Nle-CHN₂ (1.0 eq) is added HCl(g) and pyridine (5 mL).The mixture is taken up in EA and washed with 1N HCl, sat. NaHCO₃, sat.NaCl, dried (MgSO₄), filtered and concentrated to give the chloromethylketone.

To a solution of the chloromethyl ketone (1.0 eq) in anhydrous THF underAr at R.T. is added furfuryl mercaptan (2.0 eq) and Et₃ N (2.0 eq). Thereaction mixture is stirred at R.T. for 16 h, and extracted with EA. Thecombined organic layers are washed with sat. NaCl, sat. NaHCO₃, 1N HCl,dried (MgSO₄), filtered and concentrated. Purification by flashchromatography on silica gel affords the furfurylthio-derivative. Thederivative is treated with 4N HCl/dioxane to yield the hydrochloridesalt.

To a solution of the above salt (1.0 eq) in anhydrous CH₂ Cl₂ at R.T.under Ar is added HOBT (2.0 eq), EDC (1.0 eq), Et₃ N (1.0 eq) andN-Cbz-L-Leu-L-Leu-OH (1.1 eq). After 6 h the reaction mixture is washedwith sat. NaHCO₃, 1N HCl, sat. NaCl, dried (MgSO₄) filtered andconcentrated. Purification by flash chromatography on silica gel givesthe title compound.

EXAMPLE 20 Preparation of(2SR)-N- (2R)-2-(1'-phenyl-1'-propene)-4-methylpentanoyl!!-L-Leu N-2-(4-methyl-4-pentenal)!amide

To a stirred solution of 4-methylvaleric acid (10.8 mL, 86.1 mmol) inanhydrous CH₂ Cl₂ (25 mL) was added thionyl chloride (25 mL, 0.34 mmol).The mixture was placed under reflux for 24 h. Then solvent and excessthionyl chloride were removed in vacuo to give the acid chloride as anoil (10.2 g,90%). The acid chloride was used directly in the next step.

To the acid chloride (2.7 g, 20.3 mmol) in anhydrous CH₂ Cl₂ (50 mL) atR.T. under Ar was added DMAP (0.10 g), Et₃ N (4.6 mL, 33.8 mmol) and(4S, 5R)-(-)-4-methyl-5-phenyl-2-oxazolidinone (3.0 g, 16.9 mmol). Thereaction mixture was stirred for 16 h then washed with 1N HCl (2×10 mL),sat. NaHCO₃ (2×10 mL), sat. NaCl (2×10 mL), dried (MgSO₄), filtered andconcentrated. Purification by flash chromatography on silica gel (EA:H;1:20) afforded the imide as an oil (2.8 g, 61%): ¹ H NMR (CDCl₃, 300MHz) δ 0.80-1.10 (m, 9H), 1.60-1.90 (m, 3H), 2.40-2.55 (m, 2H),4.80-4.90 (m, 1H), 5.70-5.80 (m, 1H), 7.20-7.50 (m, 5H) ppm.

To a solution of the imide (2.5 g, 9.14 mmol) in anhydrous THF (40 mL)at -78° C. under Ar was slowly added a 1.5M solution of lithiumdiisodiisopropylamide (LDA) in anhydrous THF (6.0 mL, 9.14 mmol)followed by cinnamylbromide (1.8 g, 9.14 mL) in anhydrous THF (10 mL).The reaction mixture was stirred at -78° C. for 1 h then graduallywarmed to R.T. Stirring was continued at R.T. for 1 h then the mixturewas treated with 1N HCl (5 mL). The solvent was removed and the aqueouswas taken up in EA (70 mL). The aqueous was separated and the organicwas washed with sat. NaCl (2×10 mL), 1N HCl (2×10 mL), sat. NaHCO₃ (2×10mL), sat. NaCl (2×10 mL), dried (MgSO₄) filtered and concentrated.Purification by flash chromatography on silica gel (EA:H; 1:4) affordedthe alkylated derivative as an oil (3.5 g, 98.7%): ¹ H NMR (CDCl₃, 300Mhz) δ 0.73 (d, 3H, J=6.0 Hz), 0.80-0.92 (m, 6H), 1.14-1.40 (m, 1H),1.48-1.79 (m, 1H), 1.75-1.84 (m, 1H), 2.42 (m, 2H), 4.10-4.20 (m, 1H),4.71-4.78 (m, 1H), 5.28 (d, 1H, J=9.0 Hz), 6.35 (m, 1H), 6.45 (m, 1H),7.21-7.25 (m, 5H) ppm.

To a solution of the above product (0.65 g, 1.66 mmol) in 3:1 MeOH:H₂ O(20 mL) at R.T. were added LiOH.H₂ O (0.61 g, 4.98 mmol) and 30% H₂ O₂(0.83 mL). The reaction mixture was stirred for 4 h. The mixture wascooled to 0° C. and quenched by the addition of 1M Na₂ S₂ O₃ (1.6 mL)and allowed to warm to R.T. After 14 h the resulting solution was pouredonto sat. NaHCO₃ (20 mL). The aqueous was extracted with CH₂ Cl₂ (3×30mL) then acidified with 1N HCl (pH=3). The aqueous was then extractedwith CH₂ Cl₂ (3×20 mL) and the combined organics were dried (MgSO₄),filtered and concentrated. The residue was purified by flashchromatography on silica gel (EA:H; 1:1) to give(2S)-2-(1'-phenyl-1'-propene)-4-methylpentanoic acid as an oil (0.30 g,78.0%): ¹ H NMR (CDCl₃, 300 MHz) δ 0.88-1.01 (m, 6H), 1.29-1.42 (m, 3H),1.59-1.70 (m, 2H), 2.55 (m, 3H), 6.13 (m, 1H), 6.47 (d, 1H, J=6.0 Hz),7.31-7.40 (m, 5H) ppm.

To a stirred solution of L-Leu-OMe HCl (0.14 g, 0.70 mmol) and(2S)-2-(1'-phenyl-1'-propene)-4-methylpentanoic acid (0.16 g, 0.69 mmol)in anhydrous CH₂ Cl₂ (15 mL) at R.T. were added HOBT (0.19 g, 1.38mmol), EDC (0.15 g, 0.78 mmol) and Et₃ N (0.91 mL, 0.70 mmol). After 16h the reaction mixture was washed with sat. NaHCO₃ (2×10 mL), 1N HCl(×10 mL), sat. NaCl (2×10 mL), dried (MgSO₄), filtered and concentrated.Purification of the crude residue by flash chromatography on silica gel(EA:H; 1:1) afforded the methyl ester as an oil (0.24 g, 95%): ¹ H NMR(CDCl₃, 300 MHz) δ 0.76-0.78 (d, 3H, J=6.0 Hz), 0.82-1.06 (m, 3H),1.26-1.41 (m, 1H), 1.44-1.72 (m, 2H), 2.22-2.30 (m, 2H), 2.35-2.46 (m,1H) 3.72 (s, 3H), 4.57 (m, 1H), 6.16 (m, 1H), 6.43 (m, 1H), 7.25-7.41(m, 5H) ppm.

To the above methyl ester (0.28 g, 0.78 mmol) in MeOH-H₂ O (15 mL-5 mL)with stirring at 0° C. was added 1N LiOH.H₂ O (0.95 mL) and 30% H₂ O₂(1.4 mL). After 2 h 1N HCl (4 mL) was added and the aqueous layer wasextracted with EA (3×30 mL). The combined organics were washed with sat.NaCl (2×20 mL), dried (MgSO₄), filtered and concentrated to give a cruderesidue. Purification by flash chromatography on silica gel (EA:H; 1:1)afforded the acid as an oil (0.21g, 0.61 mmol): ¹ H NMR (CDCl₃, 300 MHz)δ 0.59-1.10 (m, 12H), 1.12-1.71 (m, 6H), 2.60-2.63 (m, 3H), 4.45 (m,1H), 6.21 (m, 1H) 6.40 (m, 1H), 7.25-7.41 (m, 5H) ppm.

To a stirred solution of the acid (0.25 g, 0.72 mmol) and ethyl2-amino-4-methyl-4-pentenoate (0.16 g, 0.8 mmol) in anhydrous CH₂ Cl₂(15 mL) at R.T. were added HOBT (0.19 g, 1.44 mmol), EDC (0.15 g, 0.79mmol) and Et₃ N (0.12 mL, 0.80 mmol). After 4 h the reaction mixture waswashed with sat. NaHCO₃ (2×10 mL), 1N HCl (2×10 mL), sat. NaCl (2×10mL), dried (MgSO₄), filtered and concentrated. The residue was purifiedby flash chromatography on silica gel (EA:H; 1:1 ) to give (2SR)- (2R)-2-(1'-phenyl-1'-propene)-4-methyl pentanoyl!!-L-Leu N- 2-(ethyl4-methyl-4-pentenoate)!amide as a solid (0.18 g, 51%): ¹ H NMR CDCl₃,300 MHz) δ 0.81-1.10 (m, 12H), 1.30-1.73 (m, 15H), 2.27-2.51 (m, 2H),4.14-4.17 (m, 2H), 4.22-4.49 (m, 2H), 4.59 (m, 1H), 6.21 (m, 1H), 6.43(m, 1H), 7.17-7.19 9m, 2H), 7.25-7.41 (m, 5H) ppm.

To the above ethyl ester (0.3 g, 0.62 mmol) in anhydrous THF (20 mL) at0° C. under Ar with stirring was added LiBH₄ (47 mg, 2.15 mmol). After30 min at 0° C. the reaction mixture was warmed to R.T. Stirring wascontinued for 2 h then quenched with 1N HCl (2 mL). The mixture wasextracted with EA (3×10 mL). The combined organics were washed with sat.NaHCO₃ (2×10 mL), 1N HCl (2×10 mL), sat. NaCl (2×10 mL), dried (MgSO₄),filtered and concentrated. Purification by flash chromatography onsilica gel (EA) gave the alcohol as an oil (0.24 g, 87%): ¹ H NMR(CDCl₃, 300 MHz) δ 0.72-1.02 (m, 12H), 1.20-1.86 (m, 13H), 2.21-2.56 (m,4H), 3.60-3.68 (m; 2H), 4.21-4.25 (m, 1H), 4.80-4.85 (m 1H), 6.10-6.15(m, 1H), 6.45-6.50 (m, 1H), 7.17-7.19 (m, 2H), 7.25-7.35 (m, 5H) ppm.

To anhydrous DMSO (0.08 mL, 1.08 mmol) in anhydrous CH₂ Cl₂ (5 mL) at-78° C. under Ar with stirring was added oxalyl chloride (0.06 mL, 0.68mmol). The mixture was stirred for 30 min then the alcohol (0.19 g, 0.45mmol) in anhydrous CH₂ Cl₂ (5 mL) was added. Stirring was continued at-78° C. for 2 h. Et₃ N (0.29 mL) was then added to the reaction mixtureand the mixture was allowed to gradually warm to R.T. Stirring wascontinued for 1 h. The reaction mixture was taken up in additional CH₂Cl₂ (20 mL). The organic was washed with sat. NaHCO₃ (2×10 mL), 1N HCl(2×10 mL), sat. NaCl (2×10 mL), dried (MgSO₄), filtered and concentratedto give a crude residue. Purification by flash chromatography on silicagel (EA:H; 1:1) afforded the title compound as an oil (20.0 g, 10%):Reporting a mixture of diastereomers ¹ H NMR (CDCl₃, 300 MHz) δ0.78-1.79 (ol-m, 23H), 1.96-2.66 (ol-m, 2H), 4.11-4.93 (ol-m, 4H),5.77-6.43 (ol-m, 3H), 7.19-7.25 (ol-m, 5H), 7.97-8.06 (ol-m, 2H),9.56+9.57 (s+s, 1H) ppm.

EXAMPLE 21 Preparation of N-Cbz-L-Pro-L-Leu-L-norleucinal

The title compound was isolated as a colorless oil (0.13 g) using themethodology outlined in Example 2: ¹ H NMR (CDCl₃, 300 MHz) δ 0.80-0.91(m, 9H), 1.24-2.22 (m, 3H), 3.45-3.60 (m, 2H), 4.11-4.43 (m, 3H),5.06-5.16 (m, 2H), 6.40-7.00 (m, 2H), 7.30-7.40 (m, 5H), 9.5 (s, 1H)ppm.

EXAMPLE 22 Preparation of N-Cbz-L-Leu-L-Leu-DL-cyclohexylglycinal

Using the procedure set forth in Example 3 the title compound wasisolated as a yellowish solid (50.0 mg): Reporting a mixture ofdiastereomers ¹ H NMR (CDCl₃, 300 MHz) δ 0.87-1.72 (ol-m, 29H),1.99+2.00 (s+s, 3H), 4.35-4.75 (ol-m, 3H), 6.57-7.82 (ol-m, 3H),9.56-9.63 (ol-m, 1H) ppm.

EXAMPLE 23 Preparation of N-Fmoc-L-Leu-L-Leu-DL-norleucinal

The title compound was prepared from the corresponding propane thiolester using substantally the same procedure described in Example 3.N-Fmoc-L-Leu-L-Leu-DL-norleucinal was isolated as a colorless oil (15mg): Reporting a mixture of diastereomers ¹ H NMR (CDCl₃, 300 MHz) δ0.52-0.61 (ol-m, 3H), 0.80-1.05 (ol-m, 16H), 1.22-1.26 (ol-m, 2H),2.02-2.10 (ol-m, 4H), 4.04-4.69 (ol-m, 5H), 5.21-5.27 (ol-m, 1H),7.23-7.84 (ol-m, 8H), 9.50-9.56 (ol-m, 1H) ppm.

EXAMPLE 24 Preparation of (2SR)-N-Ac-L-Leu-L-Leu N2-(trans-4-hexenal)!amide

The title compound was isolated as a white solid (0.48 g) following theprocedure set forth in Example 9: Reporting a mixture of diastereomers ¹H NMR (CDCl₃, 300 MHz) δ 0.89-1.26 (ol-m, 12H), 1.51-1.74 (ol-m, 9H),1.93 (s, 3H), 2.40-2.61 (ol-m, 2H), 3.40-4.01 (ol-m, 0.3H), 4.35-4.61(ol-m, 3H), 5.30-5.37 (ol-m, 1H), 5.49-5.50 (ol-m, 1H), 6.70-7.71 (ol-m,3H), 9.50-9.56 (ol-m, 0.7H) ppm.

EXAMPLE 25 Preparation of N-Ac-L-Leu-L-Phe-DL-norleucinal

N-Ac-L-Leu-L-Leu-DL-norleucinal was isolated as a white solid (0.12 g)using similar methodology that is set forth in Example 3: Reporting amixture of diatereomers ¹ H NMR (CDCl₃, 300 MHz) δ 0.81-0.88 (ol-m, 9H),1.23-1.83 (ol-m, 8H), 1.94-1.96 (s+s, 3H), 2.96-3.21 (ol-m, 0.4 H),3.31-3.47 (ol-m, 2H), 4.02-5.10 (ol-m, 3H), 7.05-7.20 (ol-m, 5H)9.20-9.49 (ol-m, 0.6H) ppm.

EXAMPLE 26 Preparation of N-Cbz-L-Leu-L-Leu-L-norleucinal

Using the procedure set forth in Example 2 the title compound wasisolated as a colorless oil (0.22 g): ¹ H NMR (CDCl₃, 200 MHz) δ0.86-0.93 (m, 9H), 1.23-1.86 (m, 9H), 2.37 (m, 0.2H), 3.90 (s, 2H),4.39-4.51 (m, 1H), 4.57-4.61 (m, 1H), 5.08 (m, 2H), 5.94 (hs, 1H),7.12-7.23 (m, 2H), 7.23-7.32 (m, 5H), 9.49 (s, 0.8H) ppm.

EXAMPLE 27 Preparation of (2SR)-N-Ac-L-Leu-L-Leu N-2-(4-methyl-4-pentenal)!amide

The title compound was isolated as a white solid (0.2 g) followingsubstantially the same procedure described in Example 9: Reporting amixture of diastereomers ¹ H NMR (CDCl₃, 300 MHz) δ 0.81-0.88 (ol-m, 12H), 1.31-1.90 (ol-m, 8H), 1.92 (ol-m, 3H) 2.23-2.56 (ol-m, 2H),4.70-4.80 (ol-m, 1H), 4.78-5.00 (ol-m, 3H), 7.70-7.80 (ol-m, 1H),8.01-8.60 (ol-m, 2H), 9.43+9.52 (s+s, 1H) ppm.

EXAMPLE 28 Preparation of N-Cbz-L-Leu-L-(methyl)Leu-DL-norleucinal

Following the procedures set forth in Examples 1 and 2 the titlecompound was isolated as a colorless oil (0.14 g): Reporting a mixtureof diastereomers ¹ H NMR (CDCl₃, 300 MHz) δ 0.86-1.02 (ol-m, 15H),1.26-1.98 (ol-m, 12H), 2.80-3.00 (ol-s, 3H), 4.30-4.80 (ol-m, 3H),4.95-5.20 (ol-m, 2H), 7.29-7.36 (ol-m, 5H), 9.51 (ol-s, 1H) ppm.

EXAMPLE 29 Preparation of N-Dansyl-L-Leu-L-Leu-DL-norleucinal

The title compound was isolated as a pale yellow solid (0.13 g) usingthe methodology described in Example 9: Reporting a mixture ofdiastereomers ¹ H NMR (CDCl₃, 300 MHz) δ 0.58-2.50 (ol-m, 23H), 2.89(ol-s, 6H), 3.30-3.70 (ol-m, 1H), 4.38-4.52 (ol-m, 2H), 4.81-4.9 (ol-m,1H), 5.30-5.71 (ol-m, 1H), 6.75-6.96 (ol-m, 1H), 7.19-7.36 (ol-m, 2H),7.50-7.65 (ol-m, 2H), 8.20-8.30 (ol-m, 2H), 8.56-8.61 (ol-m, 1H),9.52-9.61 (ol-s, 1H) ppm.

EXAMPLE 30 Preparation of N-Ac-L-Phe-L-Leu-DL-norleucinal

The title compound N-Ac-L-Phe-L-Leu-DL-norleucinal was obtained as awhite solid (0.19 g) following substantially the same proceduredescribed in Example 9: Reporting a fully hydrated amino-aldehyde ¹ HNMR (CDCl₃, 300 MHz) δ 0.64-1.01 (ol-m, 9H), 1.51-1.71 (ol-m, 9H),1.87-1.95 (ol-s, 3H), 2.79-3.17 (ol-m, 2H), 3.34-3.41 (ol-s, 3H),3.85-4.68 (ol-m, 4H), 7.16-7.29 (ol-m, 5H) ppm.

EXAMPLE 31 ASSAYS FOR IDENTIFICATION OF COMPOUNDS HAVING ACTIVITY ASMODULATORS OF THE PROCESSING OF APP

A. Immunoblot assay for Aβ peptide

Human glioblastoma cells (ATCC Accession No. HTB16) were stablytransfected with a DNA expression vector encoding a 695 amino acidisoform variant of the amyloid precursor protein (APP) containing thefamilial Swedish double mutations at codons 670 and 671 (K to N and M toL, respectively; see Mullan et al. (1992) Nature Genet. 1:345-347) andan additional mutation at codon 717 (V to F; see Murrell et al. (1991)Science 254:97-99) to produce cells designated HGB 717/Swed. High levelsof Aβ are detectable in the conditioned medium isolated from HGb717/Swed cultured cells. The medium also contains larger secretedfragments, α-sAPP₆₉₅, which are alternatively processed APP fragmentswhose generation precludes Aβ formation.

HGB 717/Swed cells were grown at 37° C. under a 5% carbon dioxideatmosphere in Dulbecco's modified eagle medium (DMEM; Gibco)supplemented with 10% heat-inactivated fetal calf serum, 0.45% glucose,2 mM L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycinsulfate (Gemini Bioproducts). Approximately 1×10⁶ cells were incubatedovernight in 5 ml of DMEM containing varying μM final concentrations ofdesired test compounds or a DMSO control. Conditioned medium wascollected, and unwanted cells and debris were removed by sedimentationat 3,000 rpm at 4° C.

Aβ peptides were isolated from the medium by immunoaffinity purificationusing an Aβ-specific antibody. To reduce the interaction of non-specificbinding of unrelated proteins, such as serum proteins, to the antibody,the medium was pre-treated with rabbit antisera and Protein A Sepharose(Pharmacia) for 4 hours at 4° C. The sepharose-bound material wasremoved by centrifugation at 3,000 rpm at 4° C. for 10 min, and Aβpeptides were immunoaffinity purified from the clarified medium byincubation overnight with an affinity purified polyclonal rabbitantibody (referred to as 2939) prepared against a synthetic Aβ peptidecorresponding to amino acids 1 to 28. Protein A-conjugated sepharose wasadded to immobilize the Aβ-antibody complexes, and the resin waspelleted by centrifugation at 3,000 rpm at 4° C. for 10 min. The Aβ-antibody complexes were eluted from the matrix by denaturing thecomplex by boiling in the presence of SDS.

Equal portions of each sample were loaded on 16% Tricine gels (Novex),and subjected to electrophoresis. Resolved proteins were transferredfrom the gel to Hybond nitrocellulose (Amersham, Arlington Heights,Ill.) by electroblotting, and incubated with the commercially availablemonoclonal antibody 6E10 (obtained from Drs. Kim and Wisniewski,Institute for Basic Research, New York, see, published International PCTapplication WO 90/12871), which specifically recognizes Aβ residues 1 to17. Specifically bound antibody was detected using a biotinylated goatanti-mouse IgG secondary antibody (Sigma), followed by the addition ofstreptavidin conjugated to horseradish preoxidase (Amersham, ArlingtonHeights, Ill.), and documented by luminescent detection (Amersham).Levels of Aβ peptides were determined by laser densitometry ofvisualized films. A positive result in the assay is a decrease in theformation of the 4-kDa Aβ peptides relative to the DMSO control. Thevalues for the relative percent inhibition of Aβ formation for severalof the compounds are shown in Table I:

    ______________________________________                                        INHIBITION OF Aβ FORMATION                                               COMPOUND           % INHIBITION OF Aβ                                    ______________________________________                                        Ac--L--L--Nl--(DM) 87.sup.a                                                   Cbz--L--L-- CH3!--Nl--(H)                                                                        97.sup.b                                                   Ac--L--L--Nl--(CN) 94.sup.a                                                   Cbz--L--F--Lene--(H)                                                                             69.sup.c                                                   Ac--L--L--Chg--(H) 99.sup.a                                                   Cbz--L--F--Cha--(H)                                                                              100.sup.b                                                  Ac--L--L--Cha--(H) 46.sup.c                                                   Ac--L--L--Nlene--(H)                                                                             96.sup.a                                                   Cbz--P--L--Nl--(H) 80.sup.b                                                   Cbz--L--L--Lene--(H)                                                                             95.sup.c                                                   Cbz--L-- CH3!--L--Nl--(H)                                                                        77.sup.b                                                   2S--benzoxyvaleric--L--Lene--(H)*                                                                85.sup.b                                                   Ac--L--L--Nl--(TFMK)                                                                             50.sup.a                                                   Fmoc--L--L--Nl--(H)                                                                              89.sup.d                                                   ______________________________________                                         .sup.a Inhibition of Aβ formation was determined at a concentration      of 75 μM.                                                                  .sup.b Inhibition of Aβ formation was determined at a concentration      of 40 μM.                                                                  .sup.c lnhibition of Aβ formation was determined at a concentration      of 10 μM.                                                                  .sup.d Inhibition of Aβ formation was determined at a concentration      of 50 μM.                                                                  *dipeptide                                                                    Nl norleucine                                                                 Lene 2amino-4-methyl-4-pentenoic acid (unsaturated isobutyl leu side          chain)                                                                        Nlene 2amino-4-hexenoic acid (unsaturated nbutyl Nle side chain)              (H) aldehyde                                                                  (TFMK) trifluoromethylketone                                                  (DM) diazomethylketone                                                        (CN) nitrile                                                             

B. ELISA assay for total sAPP

Human glioblastoma cells (ATCC Acession No. HTB16) were stablytransfected with a DNA expression vector encoding the 695 amino acidisoform of the amyloid precursor protein (APP₆₉₅). The resulting cellsare designated HGB695 cells. High levels of secreted proteolyticprocessed fragments of APP₆₉₅ (sAPP₆₉₅) are detectable in the culturemedium (sAPP₆₉₅).

Approximately 1×10⁵ cells were plated into 12-well dishes and were grownfor 72 hours at 37° C. under a 5% carbon dioxide atmosphere in 1 ml ofDulbecco modified eagle medium (DMEM) supplemented with 10%heat-inactiveted fetal calf serum, 0.45% glucose, and 100 units/mlpenicillin, 100 μg/ml streptomycin sulfate and 2 mM L-glutamine.Following incubation, the medium was removed and 1 ml of supplementedDMEM medium containing 5 μl of DMSO or DMSO containing the desired testcompound within a range about 5 to 100 μM (final concentration in thewell), was added to each well, and incubation was continued for 24hours. Unwanted cells and debris were removed by sedimentation at3,000×g for 10 min at room temperature. Supernatants were stored at -20°C. for analysis.

In order to determine the amount of sAPP in the supernatants, a captureantibody, such as monoclonal antibody P2-1; which recognizes an epitopelocated in the amino terminus of APP (see, e.g., U.S. Pat. No. 5,270,165) was attached to the wells of a 96-well microtiter plate byincubating the antibody in the plate for 60 min at 37° C. The plateswere washed three times with 0.3 ml of 0.1% Tween-20 inphosphate-buffered saline (PBS). The non-specific interaction ofunrelated proteins (such as serum proteins that may interfere with theanalysis) with the antibody was reduced by incubating the pre-treatedwells for 30 min at 37° C. with a solution of 0.5% casein in PBS (150μl/well). Wells were washed thoroughly with 0.1% Tween-20 in PBS priorto analysis of samples.

The conditioned medium supernatant was diluted 1:20 in 0.95 ml of 0.1%CHAPS (3- (3-cholamidopropyl)-dimethylammonio!-1-propane-sulfonate) inPBS. Supernatant samples (100 μl/well) or sAPP standards (100 μl/well)of a range about 5 to 50 ng/ml were added to the pre-treated wells andincubated for 60 min at 37° C. The supernatant was removed and eachsample well was washed as described above. A horseradish peroxidase(HRP) conjugated goat affinity purified antibody, raised againstsAPP₆₉₅, was diluted in 0.1% Tween-20 in PBS and 10% goat serum andemployed as the "signal antibody". The unbound antibody was removed bywashing, and to each well, 0.1 ml of the chromagenic substrate K-BlueSolution (Elisa Technologies, Lexington, Ky.), was added and sampleswere incubated for 15 min at ambient temperature. Reactions were stoppedby the addition of 0.1 ml of a 9.8% solution of phosphoric acid. Theoptical density of samples was measured by spectrophotometry at 450 nm.The concentration of sAPP₆₉₅ peptides in the conditioned medium wasestimated from the sAPP₆₉₅ standard curve. Samples were analyzed induplicate with the appropriate standards and reference controls i.e., aknown protease inhibitor compound, such asN-acetylleucylleucylnorleucinal of given potency and concentration!.

C. CELL LYSATE ASSAY

In this assay, the effect of compounds on the modulation of thegeneration of partially processed C-terminal Aβ-containing amyloidogenicpeptides is examined. HGB695 human glioblastoma cells were employed andgrown in 12-well dishes essentially as described in Example 31B with thefollowing modifications. The DMEM growth media were supplemented withvarying μM concentrations of compounds or DMSO control and 100 μMleupeptin and 1 μM PMA phorbol ester and were incubated with cellcultures for 16 hours and cells were grown to approximately 2.5×10⁶cells per well.

Harvested cells from each well were lysed in 100 μl of lysis buffercontaining 50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1% NP-40, 0.1% SDS and0.5% deoxycholate supplemented with 1 mM PMSF. Equal volumes of celllysates in Laemmli SDS buffer were loaded onto 16% SDS-Tricinepolyacrylamide gels (Novex) and subjected to electrophoresis. Separatedproteins were transferred to supported nitrocellulose (BioRad) byelectroblotting. Nonspecific binding of proteins to the nitrocellulosemembrane was blocked by incubating in a solution of 5% non-fat driedmilk in PBS. The nitrocellulose membrane was washed three times in PBSand then incubated in PBS containing a 1:5000 dilution of a rabbitpolyclonal antibody raised against the C-terminal 19 amino acids of APP(provided by S. Gandy, Rockefeller University, New York). Thenitrocellulose membrane was washed as described above and incubated witha secondary biotinylated goat anti-rabbit IgG antibody. Specificallybound antibody was detected using a streptavidin horseradish peroxidaseconjugate, and visualized in combination with an enhancedchemoluminescent detection kit (Amersham). Potentially amyloidogenicpeptides greater than 9 and less than 22 kDa were quantitated bydensitometric scans of developed films within the linear range asdescribed in Example 31B. A positive result for a compound in the celllysate assay is denoted by a decrease in the levels of the protein bandsgreater than 9 and less than 22 kDa relative to the appropriate controlsamples.

D. ELISA ASSAY FOR α-sAPP

Human HGB695 glioblastoma cells transfected with DNA encoding the 695amino acid isoform of APP were grown and treated with test compound orDMSO as described in Example 31B. Medium from the cultured cells wasobtained as described in Example 31B and analyzed for α-sAPP in an ELISAassay as follows. The wells of a 96-well microtiter plate were coatedwith a monoclonal antibody that specifically recognizes the aminoterminus of human sAPP (e.g., monoclonal antibody P2-1) by incubatingthe antibody in the plate for 60 min at 37° C. The plates were washedthree times with 0.3 ml of 0.1% Tween-20 in PBS. The non-specificinteraction of unrelated proteins (e.g., serum peptides that mayinterfere with the analysis) with the antibody was reduced by incubatingthe pre-treated wells with a solution of 0.5% casein in PBS for 30 minat 37° C. Wells were washed with 0.1% Tween-20 in PBS prior to analysisof media samples.

The conditioned media were diluted 1:20 in 0.95 ml of 0.1% CHAPS in PBS.Media samples (100 μl/well) or α-sAPP standards (100 μl/well) in a rangeof about 3 to 18 ng/ml were added to the wells for a 60 min incubationat 37° C. The solution was then removed and each sample well was washedas described above. A horseradish peroxidase-conjugated rabbit affinitypurified antibody raised against a synthetic peptide consisting of thefirst 15 amino acids of Aβ (referred to as antibody 3369) was diluted in0.1% Tween-20 in PBS plus 10% normal rabbit serum and added to the wellsas the signal antibody. The plates were incubated for 60 min at 37° C.and then washed to remove unbound antibody. The chromogenic substrateK-Blue Solution (Elisa Technologies, Lexington Ky.) was added to thewells (0.1 mL/well) and allowed to incubate for 15 min at ambienttemperature. The reactions were stopped by addition of 0.1 ml of a 9.8%solution of phosphoric acid. The optical density of the samples wasmeasured by spectrophotometry at 450 nm. The concentration of α-sAPP inthe media was estimated from the α-sAPP standard curve. Samples wereanalyzed in duplicate.

EXAMPLE 32 METHOD OF INDICATING ALZHEIMER'S DISEASE

Total s-APP and α-sAPP levels in cerebrospinal fluid (CSF) of normalsubjects and members of a Swedish family carrying mutations of the APPgene at codons 670 and 671 (APP_(670/671)) were measured and compared.The APP_(670/671) mutation in the Swedish family is associated with ahigh incidence of early onset Alzheimer's disease (AD). The clinicaldiagnosis of AD in the Swedish family harboring the mutation was basedon NINCDSADRDA criteria McKhann, et al. (1984) Neurology 34:939-944!.The diagnosis was confirmed by neuropathologic examination of the brainof one deceased mutation carrier Lannfelt, et al. (1994) Neurosci. Lett.168:254-256!. Cognitive functioning was assessed with the Mini MentalState Examination (MMSE) Folstein, et al. (1975) J. Physchiatry Res.12:189-198!. The presence or absence of the APP_(670/671) mutation wasdetermined by polymerase chain reaction (PCR) nucleic acid amplificationand restriction enzyme digestion according to a previously establishedprocedure Lannfelt, et al. (1993) Neurosci. Lett. 153:85-87!.

Lumbar CSF was obtained from eight normal non-carriers in the family,two presymptomatic healthy mutation carriers, and four mutation carriersclinically symptomatic for AD. CSF samples were placed on ice, eliquotedand stored at 20° C. until tested.

A. Measurement of APP Levels

Total sAPP and δ-sAPP levels in the CSF samples were quantitated using asandwich enzyme-linked immunosorbent assay (ELISA) and immunoblottingfollowed by laser-scanning densitometry, respectively.

Standards used in the assays were obtained by isolation of total sAPPand α-sAPP from medium conditioned by human neuroblastoma IMR32 cellsATCC Accession No. CCL127! or the HGB695 cells, described above inExample 31B, as follows. Conditioned medium was filtered to remove largecell debris, and sAPP was extracted by passing the media over an anionexchange column using Toyopearl DEAE 650C resin (Toso-Hass,Philadelphia, Pa.). The bound sAPP was eluted from the column using alinear gradient of 0 to 0.6M NaCl in 50 mM sodium phosphate, pH 7.5. AllsAPP-containing eluate fractions were pooled and loaded onto animmunoaffinity column containing a monoclonal antibody that specificallyrecognizes an amino-terminal epitope of human APP (for example,monoclonal antibody P2-1raised against native human PN-2) see, e.g.,U.S. Pat. No. 5,213,962! linked to Toyopearl AF-Tresyl 650M resin(Toso-Hess). Bound sAPP was eluted from the column with 0.1M sodiumcitrate, pH 2.0. To separate α-sAPP from the other soluble forms of sAPPcontained in total sAPP that do not contain at least the amino-terminalportion of Aβ, the total sAPP was loaded onto a Sepharose 4Bimmunoaffinity adsorption column containing a monoclonal antibody thatrecognizes an epitope within the first ˜17 amino acids of Aβ (forexample, monoclonal antibody 6E 10). Specifically bound α-sAPP waseluted from the column with 0.1M sodium citrate, pH 3.0. The solution pHof the purified sAPPs was adjusted to 7.2 and 1-ml aliquots were storedat -70° C.

B. Quantitation of Total sAPP

The ELISA used to quantitate total sAPP levels in CSF samples employed amonoclonal antibody, such as P2-1, discussed above, that specificallyrecognizes an amino-terminal epitope of human APP as the captureantibody. The capture antibody was attached to the wells of a 96-wellmicrotiter plate by incubating the plate with the antibody (that hadbeen diluted in PBS, pH 7.2) for 60 min at 37° C. The plates were thenthree times with 0.3 ml of 0.1% Tween-20 in PBS. The wells were alsoincubated with a solution of 0.5% casein in PBS (150 μl/well) for 30min. at 37° C.

CSF samples (100 μl diluted 1:20) or sAPP standards (100 μl) containinga range of 5 to 50 ng/ml were added to the wells and allowed to incubatefor 60 min at 37° C. Following incubation, the wells were washedthoroughly with 0.1% Tween-20 in PBS. A goat anti-human APP polyclonalantibody raised against immunopurified APP from medium conditioned bycultured IMR32 human neuroblastoma cells (American Type CultureCollection Accession No. 127) conjugated to horseradish peroxidase wasused as the signal antibody. The antibody was diluted 1:500 in PBS and10% normal goat serum, pH 7.2, containing 0.1% Tween-20, added to thewells, and incubated for 60 min at 37° C. Unbound antibody was removedby washing as described above. To detect the bound antibody, 0.1 ml ofthe chromogenic substrate K-Blue Solution (Elisa Technologies, LexingtonKy.) was added to the wells and allowed to incubate for 15 min atambient temperature. Reactions were stopped by addition of 0.1 ml of a9.8% solution of phosphoric acid. The optical density of the samples wasmeasured by spectrophotometry at 450 nm. The concentration of sAPPpeptides in the CSF sample was estimated from the standard curve.Samples were analyzed in duplicate.

Total sAPP levels were also measured using quantitative immunoblottingessentially as described below for measurement of α-sAPP, except using amonoclonal antibody raised against a recombinant APP-containing fusionprotein (e.g., 22C11 available from Boehringer Mannheim, Indianapolis,Ind.) at a concentration of 0.3 μg/ml to specifically detect sAPP andusing purified sAPP as a standard. Quantification of total sAPP byquantitative immunoblot gave a 95% correlation to quantification byELISA.

C. Quantitation of α-sAPP

For immunoblot assays of α-sAPP contained in the CSF samples, 5-10 μl ofsample and purified standard α-sAPP of known concentrations wereanalyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) under reducing conditions. Samples were diluted into Laemmlisample buffer and loaded onto 7.5% SDS-PAGE gel. After separation, theproteins were transferred to polyvinylidene difluoride membranes (PVDFImmobilon, Millipore, Bedford, Mass.) in CAPS transfer buffer (5 mM 3-cyclohexylamine!-1-propanesulfonic acid, pH 11.0, 5% (v/v) methanol).Nonspecific binding of protein to membranes was blocked with PBScontaining 5% (w/v) non-fat dried milk and then incubated for 1 hr witha monoclonal antibody (20ml of 0.2 μg/ml) directed against theamino-terminus of Aβ (e.g., 6E10), and washed three times for one mineach time in 20 ml of PBS and 0.1% Tween. Specifically bound antibodywas detected using a biotinylated goat anti-mouse secondary antibody(Sigma) and a streptavidin-peroxidase conjugate (Amersham, ArlingtonHeights, Ill.) in combination with an enhanced chemiluminescencedetection system (Amersham, Arlington Heights, Ill.). The blots wereexposed to Kodak Scientific Imaging film X-OMAT AR and developed using aKodak X-OMAT developer. Quantitation of the α-sAPP protein in the blotswas performed by laser-scanning densitometry. Developed films within thelinear range (or multiple exposures) were scanned at 50 μM pixel sizeusing a densitometer (Molecular Dynamics, Sunnyvale, Calif.), and thedata were quantified using the ImageQuaNT software system (MolecularDynamics). Quantified volumes of α-sAPP standard were used to generatestandard curves. From the standard curves, the levels of α-sAPP in ng/mlwere determined.

D. Comparison of sAPP and α-sAPP Levels in CSF of Normal Subjects andMutation Carriers

Assays of sAPP and α-sAPP levels in CSF from normal subjects and Swedishmutation carriers were performed. Mann-Whitney non-parametric statisticswere used for comparison of the data from the experimental groups.Correlations were investigated with Pearson's and Spearman's rankcorrelation coefficients. Significance levels were set at p<0.05. TheCSF of diseased carriers had lower levels of α-sAPP than the CSF samplesof non-carriers, with no overlap between the two groups (z=-2.72;p=0.007). The CSF obtained from the four AD subjects had lower levels ofα-sAPP than that of the two pre-symptomatic AD carriers. There was astrong inverse correlation between α-sAPP concentration and age in themutation carriers (R=0.94; p=0.005). In the mutation carriers, ˜25% ofthe total sAPP in CSF was α-sAPP compared to 33% in CSF of non-carriers.This was a statistically significant difference.

The results indicate that α-sAPP and the ratio of α-sAPP to total sAPPin CSF are useful markers in the detection of neurodegenerativedisorders characterized by cerebral deposition of amyloid (e.g., AD) andin monitoring the progression of such disease. Furthermore, this assaysystem can be used in monitoring therapeutic intervention of thesediseases.

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

We claim:
 1. A method of indicating neurodegenerative disorderscharacterized by deposition of cerebral amyloid, comprising detecting adecrdease in the ratio of α-sAPP to sAPP or a decrease in the amount ofα-sAPP in a sample of CSF, wherein α-sAPP is the processed APP fragmentwhose generation precludes Aβ formation and that comprises theN-terminus of APP, but lacks the C-terminus.
 2. The method of claim 1,wherein the decrease in the ratio or amount is compared to a control. 3.The method of claim 2, wherein the control is the ratio or amount in CSFfrom individuals who do not have this disorder or is a predeterminedstandard ratio or amount.
 4. The method of claim 1, wherein the amountof α-sAPP is quantitated using a sandwich enzyme-linked immunosorbentassay.
 5. The method of claim 4, wherein:a first antibody is attached toa solid support and is directed against an amino terminal epitope ofsAPP; the first antibody is contacted with a sample of human CSF to forma complex of antibody and any reacting antigens in the CSF, and thecomplex is then contacted with a second antibody that reacts with theN-terminus of Aβ present in α-sAPP; the second antibody is an anti-humansAPP antibody conjugated to the enzyme.
 6. The method of claim 5,wherein the enzyme catalyzes reaction of a chromagenic substrate,whereby cleavage of the chromagenic substrate by the enzyme of thesecond antibody conjugate results in a detectable color change.
 7. Themethod of claim 1, wherein the amount of α-sAPP is quantitated using animmunoblotting assay and laser densitometry.
 8. The method of claim 5,wherein the first antibody is a monoclonal antibody.
 9. The method ofclaim 8, wherein the first antibody is P2-1.
 10. The method of claim 5,wherein the second antibody is a polyclonal antibody.
 11. The method ofclaim 10, wherein the second antibody is rabbit antibody
 3369. 12. Themethod of claim 5, wherein the second antibody is a monoclonal antibody.13. The method of claim 12, wherein the second antibody is 6E10.
 14. Themethod of claim 5, wherein the enzyme is horseradish peroxidase.
 15. Themethod of claim 1, wherein the amount of α-sAPP in the sample iscompared to the amount of α-sAPP in a control CSF sample.
 16. The methodof claim 15, wherein the control CSF is from a non-afflicted individualor is a predetermined standard amount.
 17. The method of claim 1,wherein the amount of α-sAPP is assessed by isolating total sAPP fromthe CSF, and then identifying the portion of total sAPP that containsonly an amino terminal part of Aβ.
 18. The method of claim 1, whereinthe amount of sAPP is quantitated using a sandwich enzyme-linkedimmunosorbent assay.
 19. The method of claim 17, wherein the portion oftotal sAPP that comprises the amino terminal part of Aβ is identifiedusing a monoclonal antibody specific therefor.
 20. The method of claim17, wherein the antibody is 6E10.
 21. The method of claim 15, whereinthe disorder is Alzheimer's Disease.
 22. The method of claim 1, whereintotal sAPP is the amount of protein in the sample that contains theN-terminus of APP.